Photodiagnosis and Photodynamic Therapy (2005) 2, 223—233

REVIEW

Photodiagnosis using Raman and surface enhanced Raman scattering of bodily fluids Janelle M. Reyes-Goddard MSc, PhD ∗, Hugh Barr, Nicholas Stone Biophotonics Group, Gloucestershire Royal Hospital, Great Western Road, Gloucester, Gloucestershire GL1 3NN, UK Available online 22 August 2005 KEYWORDS Raman; SERS; Urine; Blood; Bodily fluids; Amino acids

Summary Raman spectroscopy is the measure of inelastic scatter and has been described since 1928. It is particularly useful for medical applications because the scattered radiation measured is unique for each biomolecule. The aim of this study was to review works published in Raman scattering and surface enhanced Raman scattering (SERS) on bodily fluids. The main Raman studies have concentrated on the detection of metabolites in the aqueous humour, urine and blood-based fluids and on drugs in the latter. Other bodily fluids are also studied, e.g. vitreous humour. The Raman intensity is inherently weak. SERS provides immense amplification of the Raman signal and so it can be used for the detection of small concentrations of biochemicals. This is achieved by using nano-structured metal particles. Studies have been done to describe how the amino acids of bodily fluids interact with the nano-metal substrates. Applications of SERS in the medical field include: protein identification, illicit drugs in bodily fluids and its use as a reporter in immunoassay and DNA hybridisation. For both normal Raman and SERS the experimental design has also been reviewed. It clearly shows that the choice of wavelength is important to reduce the shot noise associated with fluorescence. Most experimental parameters show that these techniques can be useful and quick in vitro tests. © 2005 Elsevier B.V. All rights reserved.

Contents Introduction ................................................................................................. Theory of Raman scattering.................................................................................. Raman spectroscopy of aqueous humour ..................................................................... Raman spectroscopy of vitreous humour ..................................................................... Raman spectroscopy of blood, blood plasma and urine....................................................... Raman spectroscopy of other bodily fluids ................................................................... ∗

Corresponding author. Tel.: +44 1452 395712; fax: +44 8454 226489. E-mail address: [email protected] (J.M. Reyes-Goddard).

1572-1000/$ — see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/S1572-1000(05)00066-9

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J.M. Reyes-Goddard et al.

Surface enhanced Raman scattering ............................................................................. Theory of surface enhanced Raman scattering................................................................... Electromagnetic effect .......................................................................................... Chemical effect ................................................................................................. Methodology of surface enhanced Raman scattering ............................................................. SERS of amino acids based solutions ............................................................................. SERS of peptides................................................................................................. SERS of larger proteins .......................................................................................... SERS application: drug detection in bodily fluids................................................................. SERS application: immunoassay.................................................................................. SERS application: double stranded DNA (ds-DNA) ................................................................ Conclusion....................................................................................................... Acknowledgements .............................................................................................. References ......................................................................................................

Introduction The primary aim of studying any bodily fluid with Raman spectroscopy is to detect biochemical anomalies compared to normal samples. With any biological samples the main issues to be addressed are: (1) samples are heterogeneous, hence monitoring the change of one or more constituents is difficult; (2) the physiological concentration of most components is of the order of mmol/L to nmol/L in bodily fluids. One of earliest paper to study a bodily fluid, lysozyme and amino acids in solution, using laser excitation, identified and proposed peak assignments for the aromatic amino acids, four peptides and the enzyme lysozyme [1]. For example the indole ring vibration of tryptophan (Trp) at 760 cm−1 and the phenyl-breathing ring of phenylalanine (Phe) at 1002 cm−1 . With regards to peptides it was shown that they produce prominent bands at 1260 and 1660 cm−1 , these are due to amides III and I, respectively. From this early paper noteworthy points for the future work on biological fluids are: the Raman output from biological samples changes with pH and the wavelength of excitation is helps to provide good results. These points, other experimental parameters and spectral analysis will be reviewed for Raman spectra of biological fluids: aqueous humour, vitreous humour, synovial fluid, urine and blood/plasma. It will also be discussed that surface enhanced Raman scattering (SERS) is a method for amplifying Raman scatter. Its use with biological fluids will also be discussed.

Theory of Raman scattering Scientists C.V. Raman and K.S. Krishnan showed that not all scattered light is elastically scattered. Their experiment showed that if non-ionising radi-

226 226 227 227 227 227 229 229 230 230 230 231 231 231

ation, with frequency
i , was incident on a set of molecules a small fraction of the scattered radiation was shifted from the incident frequency. This inelastic scatter
i ±
is known as Raman scattering. Raman scatter can be described using the classical theory of light [2,3]. All molecules vibrate and possess a characteristic polarizability (˛), where polarizability describes the moveability of the electrons within a molecule when it exposed to an applied field. If a polarizable molecule is exposed to an electric field (˚) then the induced dipole moment (P) is P = ˛˚ Considering the vibration of the atoms in the molecule, where q is displacement, and the frequency of the excitation laser (
i ), then  
 ı˛ P = ˛0 + q0 cos(2
t) ˚0 cos(2
i t) ıq Expanding P = ˛0 ˚0 cos(2
i t) +

  1 ı˛ q0 ˚ 0 2 ıq 0

× [cos 2t(
i +
) + cos 2t(
i −
)] The last equation clearly shows that scattered light will be produced. Light scattered at the same frequency of the incident laser beam,
i, is referred to as Rayleigh scatter or elastic scatter. The radiation at
i +
is referred to as anti-stokes Raman scatter and
i −
as stokes Raman scatter. Raman signal are inherently weak because the probability of inelastic scatter occurring is very low [2]. Fluorescence is different from Raman scatter. The former is the emission of energy as a molecule relaxes from an electronic state, while Raman is

Photodiagnosis using Raman and surface enhanced Raman scattering of bodily fluids the scatter after a ‘photon-molecule’ collision. In Raman spectroscopy, fluorescence is undesirable as its output is very broad and of much greater intensity. If the incident radiation wavelength is such that excitation to electronic states does not occur, then there will be little fluorescence. This means the inherently weak Raman scatter will be more identifiable.

Raman spectroscopy of aqueous humour One aim for studying ocular fluids is to monitor metabolic changes that occur with diseases like diabetes. Wang et al. established that metabolites, glucose, urea, lactate, ascorbate and pyruvate can be studied independently as well as in a mixture [4]. Originally, the lowest concentration of glucose to be measured in aqueous solution was 0.11 mol/L, and the period of acquisition was 1 h with 200 mW applied to the sample. This concentration is above that in the human aqueous humour and, at this power and period, the application is not useful for medical applications. It was established that the Raman intensity varies linearly with respect to concentration for glucose 0.0055—0.055 mol/L and 0.28—2.8 mol/L [4,5] and lactate 0.011—0.1 mol/L [5]. Glucose, urea and lactate within human aqueous humour sample were measured for 30—60 s at 20—25 mW laser power [5,6]. The aqueous humour within an intact rabbit’s eye can be differentiated from the lens and the cornea using Raman spectroscopy. This was done using a charge coupled device (CCD) rather than a photomultiplier counter. CCDs have many advantages over photomultipliers, including low read and dark noise; the ability to measure many wavelengths simultaneously and the large wavelength response. CCDs are physically more robust and hardwearing against intense radiation. Thus for these experiments 1-s scans were sufficient to produce data with an adequate signal-to-noise ratio. Laser excitation was 514.5 nm with a power of 25 mW on the sample [7]. Fluorescence at this wavelength in biological tissue can be a problem. One possible way to reduce it is by using surface enhanced Raman scattering [8,9]. A variation of the previous experiments was to change the wavelength of excitation [10] to longer wavelengths 632.8 nm (helium—neon laser) and 752 nm (argon pumped Ti—sapphire laser). The results showed an obvious improvement in spectral quality, as the background fluorescence decreased with the increase in wavelength. The results from another group confirmed that using longer wavelengths produce more desirable spectra for biolog-

225

ical samples [11]. This experiment was also able to verify that Raman intensity is inversely proportional to wavelength to the fourth power (I ∝ 1/4 ).

Raman spectroscopy of vitreous humour There is no Raman data for healthy vitreous humour, as the only means of collecting it is at vitrectomy. Comparisons have been made between vitreous samples removed from patients suffering with diabetic retinopathy, retinal detachment or macular pucker. The samples were collected, centrifuged into a pellet and tested using a near-infrared laser [12]. The results were compared with specimens of rat-tail tendon collagen and demineralised chick bone. This comparison allowed the authors to suggest that conjugated-aromatic glycation products produced peaks at 1604 and 3057 cm−1 . However, rat-tail tendon collagen and demineralised chick bone may not be the ideal model for comparison with vitreous humour. This is because the collagen in vitreous humour is mainly type II, with some types V and IX present. While the other tissues are predominantly type I collagen. In some vitreoretinal procedures silicone oil is used as a replacement and as a tamponade. It has been shown that silicone oil can be detected in vivo using confocal Raman spectroscopy [13], so its behaviour as a tamponade can be monitored.

Raman spectroscopy of blood, blood plasma and urine Aqueous humour has mainly been studied to determine the concentrations of metabolites such as glucose, urea, and lactic acid [4—11]. In whole blood, blood plasma and urine the average concentration of glucose, urea, and creatinine are 4—6 mmol/L, 2.5—6.7 mmol/L and 70—150 mmol/L, respectively. With normal Raman spectroscopy these solutes would just be detectable in distilled water. Unfortunately, blood, plasma and urine are not clear like water or ophthalmic fluids, so it is increasingly difficult to distinguish the constituents in these solutions using Raman. This is because the limit of detection increases with turbidity. For this type of application peak-to-peak ratio analysis is not sufficient and chemometrics has be used. Glucose and lactic acid can be predicted at an estimated concentration as low as 2.65 and 1.3 mmol/L, respectively, in solutions [11,14] or serum [15,16]. In blood, the minimum predictable estimated concentration is 3.6 mmol/L

226 [17]. Other components predicted individually or collectively using Raman spectroscopy in serum or blood are urea [14,16], albumin [15,16], triglycerides [15,16], cholesterol [16] and glycoprotein [18]. Absorbed drugs acetaminophen, phthalocyanine, ethanol and codeine can also be predicted at concentrations as low as 2.4, 200, 12 and 5 mmol/L, respectively [15,19]. Raman spectroscopy is also a useful tool for demonstrate the secondary structure of proteins. This was done for glycoproteins [18] and hemoglobin A [20] in blood samples. Creatinine is an end-product from muscle metabolism. It is steadily excreted in urine, and therefore, the concentration of creatinine can be used as an internal normalisation factor for the measurement of other metabolites in urine samples. Concentrations as low as 0.433 mmol/L can be predicted using Raman spectroscopy in a urine sample [21]. This is beneficial as the physiological range is 70—150 mmol/L. Urea, glucose and acetone are other metabolites that have been measured in urine using anti-stoke’s Raman spectroscopy [22]. Their limits of detection were 7.14, 0.084 and 5.5 mmol/L, respectively. Raman scatter as described earlier is the measure of inelastic scatter. Stoke’s Raman scatter is the quantity that is measured for most publications, however, when anti-stoke’s occurs there is no probability of fluorescence occurring. Therefore, while anti-stoke’s Raman peaks are very weak they are not masked by fluorescence. While this may be one method of ridding a spectrum of fluorescence, the Raman information is more useful when the stoke’s Raman can be measured without fluorescence.

Raman spectroscopy of other bodily fluids Raman spectroscopy can also be used with other bodily fluids. For example, it can be used to measure dopamine as an indirect quantification of aluminium in water, cerebrospinal fluid, urine and blood [23]. It can also be used to detect the presence of foreign bodies. In the knee polyethylene can be used to produce prosthetic devices. Polyethylene has a different spectrum from synovial fluid, thus Raman spectroscopy is a useful test to differentiate the polyethylene debris from synovial fluid [24]. Raman spectroscopy can also discriminate between the hydrophobic and hydrophilic bile acids [25]. However, it is imperative that the Raman signal is maximised for all of the preceding applications without damage to the biological tissue. The Raman detection limit and predictability from a

J.M. Reyes-Goddard et al. model is affected by the quality of the spectra. To improve signal to noise the period, power and wavelength need to be optimised. Using NIR excitation lasers, 785—1064 nm, produce less background fluorescence and are safer for the user and sample than ultraviolet (UV) lasers, 200—244 nm. At visible wavelengths, there is fluorescence from biological tissue. However, applications where Raman spectroscopy was used to identify two non-biological materials in bodily fluids the excitation wavelengths were in the range 468—488 nm [19,24]. This range of wavelengths maybe useful for three possible reasons: (1) the Raman signal from the non-biological molecules may be greater than the tissue fluorescence; (2) the molecules may resonate at these wavelengths; and (3) Raman intensity is inversely proportional to wavelength to the fourth power (I ∝ 1/4 ), so as the wavelength decreases the intensity of the non-biological Raman signal would increase significantly. Still the powers and periods quoted in these papers are not suitable for in vivo use, but useful clinical information can be obtained very quickly. However, initial experiments have shown that blood can be measured transcutaneously with and without a fibre optic probe [26,27].

Surface enhanced Raman scattering It has been shown that Raman spectroscopy is an important analytical tool for biochemistry. However, it is inherently weak. The components of some bodily fluids have a low concentration and by extension the Raman signal will be small or not detectable and would need amplification. Surface enhanced Raman scattering has been used to amplify the Raman signal in bodily fluids [28—33]. SERS has also been used to measure insect viruses [34,35], antiviral drugs [36] and bacteria found in the urinary tract [37].

Theory of surface enhanced Raman scattering In 1974, Fleischmann et al. [38] recorded increased Raman scattering from pyridine molecules after they had been adsorbed on a roughened silver electrode. It was suggested that this was due to the increase in surface area and hence increasing the number of molecules involved. In 1977, Jeanmaire and Van Duyne [39] and Albrecht and Creighton [40] independently identified the increased Raman scattering on a barely roughened silver electrode

Photodiagnosis using Raman and surface enhanced Raman scattering of bodily fluids surface. This discounted the theory that a greater surface area meant more molecules were excited. It also began years of postulations as to the theory behind SERS. From the equation P = ˛˚, if the electric field (˚) or the molecular polarizability (˛) changes then the dipole moment would change, and thus, the Raman output would change accordingly. It is thought that two effects contribute to SERS: the electromagnetic effect and the chemical effect. This is done by the molecule of interest being in close proximity to metallic nano-structures.

Electromagnetic effect Consider a polarizable molecule adsorbed onto a metallic nano-structure. The total effective electric field is due to (1) the enhancement of the laser’s field due to the presence of the metal and (2) the molecule radiating an amplified Raman field. Thus, the resultant electric field is significantly different. The most significant contributor to the electromagnetic effect comes from surface plasmons [41]. When light energy interacts with a metal the incident electric field initiates the movement of loosely bound conducting electrons within the field. The main attribute of light induced surface plasmons is a strong localised electric field [42].

Chemical effect If the polarizability of the adsorbate in the equation P = ˛˚ is altered then the Raman signal will also change. This occurs when the chemical structure or the local environment of a molecule is changed or affected. The charge transfer theory is the most accepted mechanism that describes chemical enhancement of the Raman signal. This occurs when the incident laser photon induces excites the electrons in the metal. The result is the metal’s electrons fill the adsorbates’ partially filled orbitals. With the additional electrons the adsorbate’s internal configuration is different and so the Raman polarizability and

Table 1

227

vibrational frequency change. On relaxation, photons are ‘released’ and the electrons recombine with the metal leaving the adsorbate molecules in a neutral but different vibrational state. These ‘released’ photons are inelastic scattered light radiation, i.e. Raman scatter. The converse of this process also applies, where the adsorbate’s electrons, move to fill the metal’s orbitals.

Methodology of surface enhanced Raman scattering In order for the effects to occur, the metal particles must be between 10 and 100 nm in diameter and near to the molecule of interest. There are various ways to make SERS-active substrates. The more familiar techniques used to produce the nanoparticles are listed and described in Table 1. Silver or gold colloids, thin film and electrodes are the most commonly used substrates.

SERS of amino acids based solutions From the instant SERS was discovered a host of work in the field of biomolecules and SERS has been produced. Studies include identification of nucleic acids and amino acids to applied applications. This work concentrates on amino acids and applied applications relevant to photodiagnosis. Amino acids are the building blocks of proteins and enzymes. There is a varying number of amino acid residues required to make up different proteins. For example, 129, 550 and 1320 amino acid residues make up lysozyme, serum albumin, and immunoglobulin G (IgG), respectively [43]. The resultant SERS spectrum is a reflection of the point of interaction with the metal, influenced by all the amino acids. To understand this composite spectrum, there needs to be a fundamental understanding of the interactions of individual amino acids and peptides with the metal substrates. Silver colloid is a SERS substrate that is not highly reliable because the solution has a high tendency to aggregate. In an early study, it was hypothe-

Common techniques used to make SERS active substrates.

Method

Description

Electrochemical Chemical reduction Evaporation Chemical etching Metallic coating

Metallic foils go through oxidation and reduction cycles to roughen the surface Metal colloids are produce by the reduction of silver or gold with various chemicals Metal vapours are deposited on various supports under vacuum conditions Acids are used to erode the surface of metal foils Silver/silver colloidal solutions are dried or evaporated onto different substrate bases

228 sized that chemical complexes are formed between aromatic amino acids and silver colloids that contribute to SERS. This is due to the aggregation of the silver particles after an amino acid is added. Particle aggregation occurs due to several factors, one being time delay. This means the aggregation process will continue with respect to time until the silver particles precipitate. A second is the chemical reaction between the amino acid and the silver colloid. Therefore, as the nano-particles continue to change in dimension due to the chemical interaction with the amino acids the absorption spectrum changes and the SERS spectrum changes [44—46]. Experiments have demonstrated that the spectra vary slightly depending on the different formulae of silver colloids and concentrations of the analytes used. An amino acid is larger than 10—100 nm and so only parts of the amino acid will form chemical links with the Ag+ of a substrate. It was first shown that the peaks at 900—940 cm−1 and 1350—1400 cm−1 are representative of the SERS of phenylalanine (Phe) carboxyl group (COO− ) and the metal bond [44,46]. Fig. 1 shows the spectra of Phe and tryptophan (Trp). Suh demonstrated that the COO− of different amino acids, glycine (Gly) and alanine (Ala), are also ionised on the surface of the metal [47]. The amino group (NH3 + ) can also be attached to the metal surface. As both ends of the amino acid will interact with the metal, it has been proposed that if the molecule is aligned such that its Raman polarizability z-component is perpendicular to the surface, the molecule would produce its characteristic bands and contribute significantly to the spectrum. Others confirmed that the amino acids do interact via COO− producing a band at 1390 cm−1 , though it is not conclusive that the amino group interacted with Ag+ [48]. The concluding hypothesis could be stated as aromatic amino acids bind to

J.M. Reyes-Goddard et al. the Ag+ within silver colloid via the carboxylate and amino groups [49]. Herne and Ahern contradicted the aforementioned findings, emphasizing that the COO− group does not interact with Ag+ . Their results show aromatic amino acids and peptides bind to Ag+ via the amine group only in the deprotonated form NH2 [50,51]. It suggested that such a disparity could be due to the differences in silver colloid manufacturing [52,53]. However, the difference should not be so significant as Herne’s silver colloid is similar to Creighton’s. Creighton’s silver colloid is the substrate used in all the previous studies. In these studies, stabilisers were used and it is more likely they may contribute to the difference in the results. Two comprehensive publications on SERS of amino acids and peptides were published in 1999 [54,55]. These fully described individual amino acids, peptides and proteins with silver electrodes. The spectra published in these papers may not be identical to those with silver colloid. Nevertheless, their findings support the theory that both terminals of an amino acid are adsorbed to the silver substrate. The probability of interaction for either ends of any amino acid varied, depending on the contributions made from the different side-chains. The interactions with the side chains is one way the amino acids can be identified, as each chain is individual. Aromatic amino acids readily give intense distinguishing SERS signatures as the rings of the side chain readily interact with metal substrates. The orientation and chemical state of these rings have been studied and different theories have been proposed [48,49,54,56]. Other amino acids like cysteine (Cys) and methionine (Met) have sulphur in their side chains. They also produce strong, distinctive spectra as sulphur has an affinity to silver [54,57,58]. Further examples are, glutamate (Glu), aspartate (Asp), glutamine (Gln) and

Figure 1 SERS spectra of phenylalanine and tryptophan acquired on Tollen reaction substrate using a near infrared laser.

Photodiagnosis using Raman and surface enhanced Raman scattering of bodily fluids asparagine (Asn) all have peaks assigned to the methylene (CH2 ) vibration due to their side chains in contact with the substrate [54,59]. It is suggested that the concentrations of excitatory amino acids Glu and Asp are indicators to injury in the central nervous system. Toxic concentrations are in the range of 2—5 ␮mol/L in cerebrospinal fluid (CSF). It was demonstrated that Glu in the range 0.4—5 ␮mol/L in solution was detectable using a silver colloid to produce SERS spectra [30]. Middle cerebral artery occlusion was performed to create ischaemic areas in rats’ brains. The extracellular brain fluid was measured using SERS before and after the procedure and a significant difference in Glu concentration was recorded [30].

SERS of peptides Most small peptides interact with Ag+ through the carboxylate group on the carboxyl terminus. Normally these peaks are at about 920 and 1410 cm−1 , but with different amino acids the shift and intensity would vary, indicating the probability of interaction and the orientation of the carboxylate group with the silver surface. With a dipeptide it is the amino residue that is closer to the carboxylate group that dominates the resultant spectrum [48,49,55]. However, if the amino acids residue at the NH3 + terminus is aromatic or contains sulphur then the resultant spectrum is also strongly influenced by those side chains [55]. Tripeptides and polypeptides behave in much the same way as dipeptides. Regardless of the sequence of the peptide, the residue closest to the carbonyl end will dominate the spectrum [55]. Also if Phe, Trp, Tyr, Cys or Met are present their characteristic peaks will also be identifiable. For polypeptides, amides I and III indicate whether the conformation is ␣-helix, ␤-sheet or random coil. For ␣-helix amide I bands are between 1658 and 1640 cm−1 while amide III are between 1310 and 1260 cm−1 . With ␤-sheet, bands are between 1680 and 1665 cm−1 and 1242 and 1235 cm−1 for amides I and III, respectively. Finally for random coil, 1666—1660 cm−1 are amide I and 1250—1240 cm−1 are amide III. From the presence of these peaks, the conformations of the proteins can be estimated. In all studies on amino acids and peptides, pH is important for two reasons. Firstly, it will alter the way negatively charged particles interact with positively charged metals like silver. But it has been shown that large changes in pH, e.g. 1—7 and 7—14 are required for SERS spectra to be altered [60,47]. Secondly, with silver colloids the rate of aggregation of the metal particles will change.

229

SERS of larger proteins The analysis of proteins using SERS began almost simultaneously with amino acids. Some other early investigations included studies of other pigmented proteins. Those that are of clinical interest included catecholamines neurotransmitters [61], haemoglobin [62,63], flavins [64], retinal chromophore [65], bile pigments [66] and ocular lens extracts [8,9]. In these papers, an objective was to understand the interaction of the molecules with either the electrodes or colloidal substrates. From these early papers, the nature of SERS became better understood as was the possible orientation of the molecules with the metal substrates. Some papers also provided early proof of principle that these tests may be useful diagnostic tools for example the use of electrodes to monitor glucose oxidase [64]. It was interesting to note that these molecules would have fluoresced if NRS had been used. These were good examples that SERS quenched the inherent fluorescence. It was also noted that pH will alter the SERS spectra. This is because the change in charge of the molecules alters its attraction to the metal. Like amino acids it can also alter the aggregation of metal colloids, which in turn changes the metal-molecule interaction. The tear film is of diagnostic value as its constituents vary with diseases or infection; and viral particles are also expressed in the tear film during viral conjunctivitis. It has been shown that a mixture of proteins, enzymes, metabolites and ions with molarities in the range nmol/L to mmol/L to mimic a human tear sample can also be measured using SERS [28]. The SERS spectrum of the synthetic tear film is shown in Fig. 2. It can be seen from this spectrum that if analysis has to be done on the constituents of the tear film, chemometrics testing is required. From the spectrum in Fig. 2, peaks from the amino acids can be identified. C C stretch of

Figure 2 SERS spectrum of a synthetic tear film on a Tollen reaction substrate using a near infrared laser.

230 the phenyl ring unique to Phe at 1004 cm−1 and Trp indole symmetric ring breathing at 759 cm−1 . Other identifiable peaks are C COO− stretch at 934 cm−1 and CH2 bend at 1442 cm−1 .

SERS application: drug detection in bodily fluids The use of illicit drugs is a problem in the sporting arena and socially in most countries. There are various needs for measuring drugs in the body. Urine is the bodily fluid that is most regularly examined as it is easily obtainable and drugs are excreted into it. SERS has been used to detect very low concentrations of drug in human urine samples. These include stimulants pemoline and pentylenetetrazole at concentrations of 0.14 and 0.18 mmol/L, respectively [31], and diuretic amiloride at 22 ␮mol/L. A consituent of urine is urea, which has a characteristic SERS peak at 1000 cm−1 . As with normal Raman spectroscopy, different wavelength of excitation means that probability of florescence will change. This change was clearly demonstrated by three independent SERS experiments of urine samples. The wavelengths of excitation were 488, 514 and 785 nm. At 488 nm, there was no characteristic urea peak at 1000 cm−1 [32]. This may be so because the shot noise associated with fluorescence was greater than the Raman signal and so the peak was not identifiable. At 514 nm, the peak is identifiable [29] but it is not very strong, again this could be due to fluorescence. At 785 nm, there was a very strong peak, with no fluorescence [33].

SERS application: immunoassay Immunoassaying is the measurement of a protein following targeting of antigens by specific antibodies. The first SERS immunoassay was set up on silver film impregnated with human thyroid stimulating hormone (TSH) antibody. A SERS active dye (known as a reporter) was attached to a second set of TSH antibodies. The capture was first by the antibodies on the silver film to the antigen and subsequently they attached to the antibody with the SERS-reporter. This arrangement places the reporter in close proximity to the silver and so SERS of the reporter will occur. The intensity of the SERS can therefore be used to quantify the antigen concentration [67]. This is the basis for all SERS-immunoassay experiments. Another exper-

J.M. Reyes-Goddard et al. iment changed the silver film to gold particles and tested different antigens and antibodies simultaneously. The results showed that the antigens link with their respective antibodies and this produced different SERS signals. Different SERS signals are produced because each antibody had a different reporter [68]. This is advantageous because the results are highly specific and more than one antigen-antibody interaction can be studied concurrently. This tool is clinically useful as the conventional technique will study one interaction at a time. SERS-immunoassay is also used with enzyme reaction [69,70].

SERS application: double stranded DNA (ds-DNA) Nucleic acids can be detected if they are adsorbed unto the metal nano-structures [71—78]. While direct detection is beneficial for genome sequencing, it is not ideal for routine clinical work. For example polymerase chain reaction (PCR) is a very costly process and if the number of cycles required for amplification of the DNA fragment could be minimised it would make the process more cost effective. 4 ,6-Diamidino-2-phenylindole dihydrochloride (DAPI) is an intercalating dye, which has an intense SERS signature when it is free. If it forms a DAPI-ds-DNA complex it no longer has a SERS signal. Hence, DAPI SERS intensity is inversely proportional to the quantity of ds-DNA present. This makes it a good gauge of the PCR cycles needed [79,80]. Fast sequencing of nucleic acids or characterizing of DNA fragments is done using a method of hybridisation. Hybridisation is where a targeted DNA fragment is labelled with a specific marker so that it can be recognised by a complimentary probe. Labellers include radioactive and fluorescence reporters, where the fluorescence reporters can be replaced by a SERS reporter. This has been shown to be advantageous because SERS results are less ambiguous than fluorescence. This is because the SERS’s output is a well defined peak, while the fluorescence is a broad band. The effectiveness of cresyl fast violet (CFV) as a SERS reporter in DNA hybridisation was demonstrated by Vo Dinh et al. [81,82]. Their work also extended to the human immunodeficiency virus (HIV) gag gene sequence. Where CFV was used like DAPI to quantify the number of PCR cycles required and CFV was used as the reporter for hybridisation [81,83]. Other SERS reporters that have been used for DNA hybridisation are 1 ,3 ,7 ,9 -hexachloro-6-carboxyfluorescin and rhodamine 6G [84,85].

Photodiagnosis using Raman and surface enhanced Raman scattering of bodily fluids

Conclusion Normal Raman spectroscopy and SERS are clearly useful in the identification of biochemical molecules from bodily fluids. Each can provide vital information on metabolite and drug concentrations and monitor therapeutic bodies using bodily fluids. When analysing spectra for the presence of therapeutic bodies, simple peak comparison may suffice. However, when the metabolite or drug peaks may occur in similar peak positions as the bodily fluid, analysis with chemometrics is required. Chemometrics requires spectra with minimal noise. This can be achieved, with the correct selection of wavelength of excitation, power of the laser and period of exposure, while taking into consideration the nature of the biological samples. While Raman is inherently weak, SERS can amplify signals such that the detection limits for various applications are well within useful clinical physiological range. SERS is an important tool both in direct and indirect applications. For direct applications, it can be used in conjunction with chemometrics to identify proteins in the tear film, which is advantageous as a near-patient diagnostic tool. Indirectly, it is an efficient and safe immunoassay and ds-DNA reporter. However, further studies need to be published to reinforce that SERS is selective and reproducible. This will enforce SERS as a clinical tool for a myriad of applications.

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

[17]

Acknowledgements

[18]

Grant support: Royal Blind Asylum & School and the Scottish National Institution for the War Blinded.

[19]

[20]

References [1] Lord RC, Yu N-T. Laser-excited Raman spectroscopy of biomolecules I. Native lysozyme and its constituent amino acids. J Mol Biol 1970;50:509—24. [2] Stevenson CL, Vo Dinh T. Signal expression in Raman spectroscopy. In: Laserna JJ, editor. Modern technique in Raman spectroscopy. Chichester: Wiley & Sons; 1996. p. 1—39. [3] Banwell CN. Raman spectroscopy. In: Banwell CN, editor. Fundamentals of molecular spectroscopy. London: McGrawHill Book Company; 1983. p. 124—54. [4] Wang SY, Hasty CE, Watson PA, Wicksted JP, Stith RD, March WF. Analysis of metabolites in aqueous solutions using laser Raman spectroscopy. Appl Optics 1993;32(6):925—9. [5] Wicksted JP, Erckens RJ, Motamedi M, March W. Monitoring of aqueous humor metabolites using Raman spectroscopy. SPIE 1994;2135:264—74. [6] Erckens RJ, Motamedi M, March WF, Wicksted JP. Raman spectroscopy for non-invasive characterization of ocular

[21]

[22]

[23]

[24]

[25]

231

tissue: potential for detection of biological molecules. J Raman Spectrosc 1997;28:293—9. Jongsma FHM, Erckens RJ, Wicksted JP, et al. Confocal Raman spectroscopy system for non-contact scanning of ocular tissues: an in vitro study. Opt Eng 1997;36(11):3191—9. Nie S, Castillo C, Berbauer K, Kuck JFR, Nabiev IR, Yu NT. Surface-enhanced Raman analysis of eye lens pigments. Appl Spectrosc 1990;44:5471—575. Sokolov K, Lutsenko S, Nabiev I, Nie S, Yu NT. Surfaceenhanced Raman analysis of biomedical eye lens extracts. Appl Spectrosc 1991;45(7):1143—8. Wicksted JP, Erckens RJ, Motamedi M, March WF. Raman spectroscopy studies of metabolites concentrations in aqueous solutions and aqueous humor specimens. Appl Spectrosc 1995;49(7):987—93. Berger AJ, Wang Y, Feld MS. Rapid, non-invasive concentration measurements of aqueous biological analytes by near-infrared Raman spectroscopy. Appl Optics 1996;35(1):209—12. Sebag J, Nie SM, Reiser K, Charles MA, Yu NT. Raman spectroscopy of human vitreous in proliferative diabetic retinopathy. Invest Ophth Vis Sci 1994;35(7):2976—80. Erckens RJ, Hosseini K, March WF, et al. Raman spectroscopy: non-invasive determination of silicone oil in the eye: potential application for intraocular determination of biomaterials. Retin J Ret Vit Dis 2002;22(6):796—9. Goetz MJ, Cote GL, Erckens R, March W, Motamedi M. Application of a multivariate technique to Raman spectra for quantification of body chemicals. IEEE Trans Biomed Eng 1995;42(7):728—31. Qu JY, Wilson BC, Suria D. Concentration measurements of multiple analytes in human sera by near-infrared laser Raman spectroscopy. Appl Optics 1999;38(25):5491—8. Berger AJ, Koo TW, Itzkan I, Horowitz G, Feld MS. Mulitcomponent blood analysis by near-infrared Raman spectroscopy. Appl Optics 1999;38(13):2916—26. Berger AJ, Itzkan I, Feld MS. Feasibility of measuring blood glucose concentration by near-infrared Raman spectroscopy. Spectrochim Acta A 1997;53:287—92. Kopecky V, Ettrich R, Hofbauerava K, Baumruk V. Structure of human ␣1 -acid glycoprotein and its high-affinity binding sites. Biochem Biophys Res Commun 2003;300:41—6. Abramczyk H, Szymczyk I. Aggregation of phthalocycnine derivatives in liquid solutions and human blood. J Mol Liq 2004;110:51—6. Ozaki Y, Mizuno A, Sato H, Kawauchi K, Muraishi S. Biomedical application of near infrared Fourier transform Raman spectroscopy. Part I: The 1064-nm excited Raman spectra of blood and Met hemoglobin. Appl Spectrosc 1992;46(3):533—6. McMurdy JW, Berger AJ. Raman spectroscopy-based creatinine measurement in urine samples from a multipatient population. Appl Spectrosc 2003;57(5):522—5. Dou X, Yamaguchi Y, Yamamoto H, Doi S, Ozaki Y. Quantitative analysis of metabolites in urine by anti-stoke’s Raman spectrosocpy. Vib Spectrosc 1997;14(2):199—205. Zhang F, Shuping B, Lui J, et al. Applications of dopamine as an electroactive ligand for the determination of aluminium in biological fluids. Anal Sci 2002;18:293—9. Visentin M, Stea S, Squarzoni S, Antonietti B, Reggiani M, Toni A. A new method for isolation of polyethylene waer debris from tissue and synovial fluid. Biomaterials 2004;25(24):5531—7. Lamcharfi L, Meyer C, Lutton C. Rationalization of the relative hydrophobicity of some common bile acid by infrared and Raman spectroscopy. Biospectroscopy 1997;3:393—401.

232 [26] Chaiken J, Finney WF, Peterson CM, et al. Non-invasive, in vivo, tissue modulated near infrared vibrational spectroscopic study of mobile and static tissues: blood chemistry. Proc SPIE 2000;3918:135—43. [27] Pilotto S, Pacheco MTT, Silveira L, Villaverde AB, Zangaro RA. Analysis of near-infrared Raman spectroscopy as a new technique for a transcutaneous non invasive diagnosis of blood components. Laser Med Sci 2001;16:2—9. [28] Reyes-Goddard JM, Woodman AC, Stone N, et al. Surface enhanced Raman scattering of the tear film. Proc SPIE 2003;5141:219—29. [29] Trachta G, Schwarze B, Sagmuller B, Brehm G, Schneider S. Combination of high-perofrmance liquid chromatography and SERS detection applied to the analysis of drugs in human blood and urine. J Mol Struct 2004;693:175—85. [30] O’Neal P, Cote GL, Motamedi M, Chen J, Lin WC. Feasibility study using surface-enhanced Raman spectroscopy for the quantitative detection of excitatory amino acids. J Biomed Opt 2003;8(1):33—9. [31] Ruperez A, Montes R, Laserna JJ. Identification of stimulant drugs by surface-enhanced Raman spectrometry on colloidal silver. Vib Spectrosc 1991;2:145—54. [32] Clavo N, Montes R, Laserna JJ. Surface-enhanced Raman spectrometry of amiloride on colloidal silver. Anal Chim Acta 1993;280:263—8. [33] Premasiri WR, Clarke RH, Womble NE. Urine analysis by laser Raman spectroscopy. Laser Surg Med 2001;28:330—4. [34] Bao PD, Liu XM, Hauang TQ, Bing J, Wu GF. Application of surface-enhanced Raman scattering spectroscopy to the study of insect virus virion. Proc SPIE 1999;3784:370—7. [35] Bao PD, Huang TQ, Liu XM, Wu TQ. Surface-enhanced Raman spectroscopy of insect nuclear polyhedrosis virus. J Raman Spectrosc 2001;32:227—30. [36] Stanicova J, Kovalcik P, Chinsky L, Miskovsky P. Preresonance Raman and surface-enhanced Raman spectroscopy study of the complex of the antiviral and antiParkinson drug anantadine with histidine. Vib Spectrosc 2001;25:41—51. [37] Jarvis RM, Goodacre R. Discrimination of bacteria using surface-enhanced Raman spectroscopy. Anal Chem 2004;76(1):40—7. [38] Fleischmann M, Hendra PJ, McQuillan AJ. Raman spectra of pyridine adsorbed at a silver electrode. Chem Phys Lett 1974;26(2):163—6. [39] Jeanmaire DJ, Van Duyne RP. Surface Raman spectroelectrochemistry. Part I. Hetercyclic aromatic and aliphatic amines adsorbed on the anodised silver electrode. J Electroanal Chem 1977;84(1):1—20. [40] Albrecht MG, Creighton JA. Anomalously intense Raman spectra of pyridine at a silver electrode. J Am Chem Soc 1977;99(15):5215—7. [41] Ruperez A, Laserna JJ. Surface-enhanced Raman spectroscopy. In: Laserna JJ, editor. Modern technique in Raman spectroscopy. Chichester: Wiley & Sons; 1996. p. 227—64. [42] Moskovits M. Surface-enhanced spectroscopy. Rev Mod Phys 1985;57(3):783—826. [43] Lehninger AL, Nelson DL, Cox MM. Nucleotides and nucleic acids. In: Lehninger AL, Nelson DL, Cox MM, editors. Principles of biochemistry. New York: Worth Publishers; 1993. p. 134—59. [44] Nabiev IR, Savchenko VA, Efemov ES. Surface-enhanced Raman spectra of aromatic amino acids and proteins adsorbed by silver hydrosols. J Raman Spectrosc 1983;14(6):375—9. [45] Picquart M, Lacrampe G, Jaffrain M. Surface enhanced Raman scattering of phenylalanine and histidine adsorbed by silver colloid. In Alix AJP, Bernard L, Manfait M, editors.

J.M. Reyes-Goddard et al.

[46]

[47]

[48]

[49]

[50]

[51]

[52]

[53]

[54]

[55]

[56]

[57]

[58]

[59]

[60]

[61]

[62]

[63]

[64]

Spectroscopy of biological molecules. Proceedings of the First European Conference on the spectroscopy of biological molecule. Chichester: John Wiley & Sons; 1986. p. 190—2. Nabiyev IR, Chumanov GD. Surface-enhanced Ramanspectroscopy of biopolymers: water soluble proteins, amino acids and dipeptides adsorbed on silver electrodes and silver hydrosols. Biofizika+ 1986;31(2):183—90. Suh JS, Moskovits M. Surface enhanced Raman spectroscopy of amino acids and nucleotides bases adsorbed on silver. J Am Chem Soc 1986;108(6):4711—8. Kim SK, Kim MS, Suh SW. Surface enhanced Raman scattering (SERS) of aromatic amino acids and their glycyl dipeptides in silver sol. J Raman Spectrosc 1987;18:171—5. Lee HI, Kim MS, Suh SW. Raman spectroscopy of lphenylalanine, l-tyrosine and their peptides adsorbed on silver surface. B Kor Chem Soc 1988;9(4):218—23. Herne TM, Ahern AM, Garrell RL. Surface enhanced Raman spectroscopy of peptides: preferential N-terminal adsorption on colloidal silver. J Am Chem Soc 1991;113:846—54. Herne TM, Ahern AM, Garrell RL. Surface-enhanced Raman spectroscopy of tripeptides adsorbed on colloidal silver. Anal Chim Acta 1991;246:75—84. Ahern AM, Garrell RL. Characterization of polacrylamide gel formation and structure by surface-enhanced Raman spectroscopy. Langmuir 1988;4:1162—8. Ahern A, Garrell RL. In situ photoreduced silver nitrate as substrate for surface enhanced Raman spectroscopy. Anal Chem 1997;59:2813—6. Stewart S, Fredericks PM. Surface-enhanced Raman spectroscopy of peptides and proteins adsorbed on an electrochemically prepared silver surface. Spectrochim Acta A 1999;55:1615—40. Stewart S, Fredericks PM. Surface Raman spectroscopy of amino acids adsorbed on an electrochemically prepared silver surface. Spectrochim Acta A 1999;55:1641—60. Lee HI, Suh SW, Kim MS. Raman spectroscopy of ltryptophan containing peptides adsorbed on a silver surface. J Raman Spectrosc 1988;9:491—5. Watanabe T, Maeda H. Adsorption-controlled redox activity. Surface-enhanced Raman investigation of cystine versus cysteine on silver electrodes. J Phys Chem US 1989;93:3258—60. Lee H, Kim MS, Suh SW. Raman spectroscopy of sulphur containing amino acids and their derivatives adsorbed on silver. J Raman Spectrosc 1991;22:91—6. Chumanov GD, Efremov RG, Nabiev IR. Surface-enhanced Raman spectroscopy of biomolecules. Part 1: Water soluble proteins, dipeptides and amino acids. J Raman Spectrosc 1990;21:43—8. Arenas JF, Castro JL, Otero JC, Marcos JL. Study of interaction between aspartic acid and silver by surfaceenhanced Raman scattering on H2 O and D2 O sols. Biopolymers 2001;62:241—8. Lee NS, Hsieh YZ, Paisley RF, Morris MD. Surface-enhanced Raman spectroscopy of catecholamines neurotransmitters and related compounds. Anal Chem 1988;60(5):442—6. Smulevich G, Spiro TG. Surface enhanced Raman spectroscopic evidence that adsorption on silver particles can denature heme-proteins. J Phys Chem US 1985;89(24):5168—73. de Groot J, Hester RE. Surface-enhanced resonance Ramanspectroscopy of oxyhemoglobin adsorbed onto colloidal silver. J Phys Chem US 1987;91(7):1693—6. Holt RE, Cotton TM. Surface-enhanced resonance Raman and electrochemical investigation of glucose oxidase catalysis at a silver electrode. J Am Chem Soc 1989;111:2815—21.

Photodiagnosis using Raman and surface enhanced Raman scattering of bodily fluids [65] Huang JY, Lewis A, Loew L. Absorption and vibrational spectra of the surface properties of molecular monolayers with large light-induced dipole alterations. Spectrochim Acta A 1988;44(8):793—803. [66] Hsieh YZ, Lee NS, Sheen RS, Morris M. Surface-enhanced Raman spectroscopy of free and complexed bilirubin. Langmuir 1987;3(6):1141—6. [67] Rohr TE, Cotton TM, Fan N, Tarcha PJ. Immunoassay employing surface-enhanced Raman spectroscopy. Anal Biochem 1989;182:388—98. [68] Ni J, Lipert RJ, Dawson GB, Porter MD. Immunoassay readout method using extrinsic Raman labels adsorbed on immuno-gold colloid. Anal Chem 1999;71:4903—8. [69] Dou X, Takama T, Yamagauchi Y, Ozaki Y. Enzyme Immunoassay utilizing surface-enhanced Raman scattering of the enzyme reaction product. Anal Chem 1997;69:1492—5. [70] Hawi SR, Rochanakij S, Adar F, Campbell WB, Nithipatikom K. Detection of membrane-bound enzyme in cells using immunoassay an Raman microspectroscopy. Anal Biochem 1998;259:212—7. [71] Koglin E, Sequaris JM, Valenta P. Surface Raman spectra of nucleic acid components adsorbed at a silver electrode. J Mol Struct 1980;60:412—25. [72] Ervin KM, Koglin E, Sequaris JM, Valenta P, Nurnburg HW. Surface enhanced Raman spectra of nucleic acid components adsorbed at a silver electrode. J Electroanal Chem 1980;114:179—94. [73] Brabec V, Nike K. Raman scattering from nucleic acids adsorbed at a silver electrode. Biophys Chem 1985;23:63—70. [74] Koglin E, Sequaris JM, Valenta P. Surface enhanced Raman spectroscopy of nucleic acid bases on Ag electrodes. J Mol Struct 1982;79:185—9.

233

[75] Watanabe T, Kawanami O, Katoh H, Honda K, Nishimura Y, Tsuboi M. SERS study of molecular adsorption: some nucleic acid bases on Ag electrode. Surf Sci 1985;158:341—51. [76] Koglin E, Sequaris JM, Fritz JC, Valenta P. Surface enhanced Raman scattering (SERS) of nucleic acid bases adsorbed on silver colloid. J Mol Struct 1984;114:219—23. [77] Kneipp H, Flemming J. Surface enhanced Raman scattering (SERS) of nucleic acids adsorbed on colloidal silver particles. J Mol Struct 1986;145:173—9. [78] Sequaris JM, Koglin E, Valenta P, Nurnberg HW. Surfaceenhanced Raman scattering (SERS) spectroscopy of nucleic acids. Ber Bunsen Phys Chem 1981;85(5):512—3. [79] Dou X, Takama T, Yamaguchi Y, et al. Quantitative analysis of double-stranded DNA amplified by a polymerase chain reaction employing surface-enhanced Raman spectroscopy. Appl Optics 1998;37:759—63. [80] Dou X, Ozaki Y. Surface-enhanced Raman scattering of biological molecules on metal colloids: basic studies and applications to quantitative assay. Rev Anal Chem 1999;18(4):285—321. [81] Vo Dinh T. Surface enhanced Raman spectroscopy using metallic nanostructures. Trac-Trend Anal Chem 1998;17(8—9):557—82. [82] Vo Dinh T, Houck K, Stokes DL. Surface enhanced Raman geneprobes. Anal Chem 1994;66(20):3379—83. [83] Narayana I, Stokes D, Vo-Dinh T. Surface-enhanced Raman gene probe for HIV detection. Anal Chem 1998;70:1352—6. [84] Graham D, Mallinder BJ, Smith WE. Detection and identification of labelled DNA by surface enhanced resonance Raman scattering. Biopolymers 2000;57:85—91. [85] Docherty FT, Clark M, Mc Nay G, Graham D, Smith WE. Multiple labelled nanoparticles for biodetection. Faraday Discuss 2004;126:281—8.