History Of Surface Enhanced Raman Spectroscopy

Published Date: 02 Nov 2017

Introduction and Literature Review

Analgesics are a type of drug that is used to relieve pain temporarily. Analgesics work by blocking pain signals by slowing down the central nervous system and by changing the way in which the brain interprets these signals. The majority of analgesics are safe to use if they are taken as prescribed or instructed by your doctor or pharmacist, and in accordance with the manufacturer’s instructions on the packaging. Some additional precautions may need to be followed for patients with pre-existing medical conditions, such as kidney failure or gastric ulcers (Australian Drug Foundation 2011). There are two main types of analgesics: non-narcotic and narcotic. Non-narcotic analgesics, also called painkillers, are used to reduce pain such as headaches, colds, flu symptoms, muscular pain, arthritis and other minor conditions. These do not usually require a prescription to obtain them and can be purchased over-the-counter (OTC), and these can be in capsule, tablet or powdered form. Narcotic analgesics, also called opoid medications, are used for relief of severe or chronic pain. Narcotic analgesics can also be used for control of coughs (codeine, hydrocodone) and also in the treatment of addiction to other opioids such as Methadone. These can only be obtained with a prescription from a doctor or health professional. Narcotic analgesics can be taken orally in tablet or capsule form, or injected. They are also available as a skin patch, syrup, liquid and as suppositories. Non-narcotic analgesics are not usually addictive. When narcotic analgesics are used infrequently and under the guidance of a physician, they can be a safe and effective form of pain relief. But regular use of narcotic analgesics can be psychologically and physically addictive.

It is not illegal to use narcotic analgesics (opioids) when prescribed by a physician, but it is illegal to sell or obtain narcotic analgesics from an unauthorised person under The Misuse of Drugs Act 1977, the Misuse of Drugs Act 1984 and the Criminal Justice (Psychoactive Substances) Act 2010, which are the acts of the Oireachtas regulating drugs in Ireland. Possessing and/or selling narcotic analgesics for the purpose of trafficking is a criminal offense. For this reason, the detection of analgesics in substances such as liquids has become significant, and the availability of low cost, user-friendly and easily accessible instrumentation in the detection of such drugs is imperative (Irish Focal Point, 2011).

There are many different chromatographic techniques employed in the detection of analgesics in liquids such as; liquid chromatography (LC) and high performance liquid chromatography (HPLC) using detectors such as UV-Vis, diode-array (DAD) or fluorimetric detector; liquid chromatography with tandem mass spectrometry (LC-MS-MS) and gas chromatography with mass spectrometry (GC-MS), which enable the determination of such compounds down to the ng/l level.

This study will examine the principle and application of an enhanced form of Raman spectroscopy called Surface Enhanced Raman spectroscopy (SERS) in the detection of analgesics in solution, and the theory underlying this vibrational technique. The study will also look at the types of instrumentation used in the characterisation of nanoparticles, in particular copper nanoparticles. SERS has been employed for many years since its accidental discovery in 1974, using metals such as silver or gold, but due to the high cost of such metals, the use of copper nanoparticles in this field is seen to be a good low cost alternative.

History of Surface Enhanced Raman Spectroscopy (SERS)

The SERS phenomenon was discovered accidentally, and was first demonstrated in 1974 by Martin Fleischmann and his colleagues while they tried to do Raman on pyridine, that was adsorbed on a silver surface roughened by an electrochemical method (Rzepka, 2012). The original idea was to generate a high surface area on the roughened metal. It was gradually realised that surface area was not the key point in this phenomenon. In 1977 progress was made, when different groups studying the effect, found the rough silver electrode produced a Raman spectrum that was 106 times more intense than what was expected. This extremely powerful signal was the first appearance of SERS. More recently the SERS effect has triumphed over the disadvantage of the small cross section of Raman spectroscopy and can be applied when studying single molecule spectroscopy (Jiang, 2004).

SERS not only gives way to exciting opportunities in the biophysical and biomedical spectroscopy field where it provides ultra-sensitive detection and characterisation of biophysically/biomedically relevant molecules and processes, but also as vibrational spectroscopy with very high spatial resolution i.e. the detection of small molecules (Rzepka, 2012). 

1.3 Nanotechnology

Nanotechnology deals with molecules that are approximately between 1 - 100 nanometres in size. The term "nanotechnology" has evolved over the years to mean "anything smaller than microtechnology", things that are nanoscale in size, such as nano powders. (Nanotechnology Now, 2011). Nanotechnology is a collection of different technologies and approaches, which all use the physical properties of particles on the nanometre scale. There are many areas of study in nanotechnology such as; nanomaterials and nanoelectronics, nanobiotechnology and nanomedicine, nanotools, nanoinstruments and nanodevices. Nanomaterials are expected to have a major influence in the future on practically all fields in which materials play a role. They include ultra-thin coatings and active surfaces as well as the new applications of chemical engineering. Nanoelectronics have enhanced the areas of information and communication technologies by continuing or overcoming (with the aid of quantum electronics) Moore’s law of doubling data storage and processing capacities every 18 months. Nanobiotechnology will make an improved difference in medicine, for pharmaceuticals and diagnostics, in innumerable industrial processes, agriculture and food industry. Nanotools are nanotech enabling technologies, such as electron microscopes, Scanning Tunnel Microscope (STM), Atomic Force Microscope (AFM) and ultra-precision machines (EUROPA, 2006).

1.4 Synthesis Routes for Copper Nanoparticles

Compared to other metals, such as silver and gold, synthesising copper nanoparticles is not as unproblematic, as copper nanoparticles in aqueous solutions are not stable. Surface oxidation occurs when the copper nanoparticles are exposed to air and they begin to aggregate over a short time. In order to avoid oxidation the reduction methods are executed under an inert atmosphere of argon or nitrogen, in the presence of protective polymers or surfactants, in organic solvents or microemulsion systems.

Copper nanoparticles can by synthesised using Chemical, Physical and Biological Methods. Chemical Methods include; chemical reduction method, microemulsion/colloidal method, sonochemical method, microwave method, electrochemical method, solvothermal decomposition. Physical Methods include; pulse laser ablation/deposition, mechanical/ball milling method, pulsed wire discharge method (Umer et al. 2012).

The chemical reduction method uses a copper salt which is reduced using a reducing agent such as ascorbate (vitamin C), glucose, hydrazine (N2H2), polyol, sodium borohydride (NaBH4) or isopropyl alcohol (C3H8O) with cetyltrimethylammonium bromide (CTAB) (Bahadory, 2008)

1.5 Instrumentation for Characterisation

In this section I will describe the instrumentation used to characterise the copper nanoparticles.

1.5.1 Ultraviolet/Visible Spectroscopy

UV/Vis spectroscopy employs a beam of light from a visible or UV light source that is separated into its component wavelengths by a diffraction grating or prism (Figure 1). Each single wavelength is split into two beams of equal intensity by a rotating disc or split mirror. One beam; the sample beam, passes through a cuvette containing the sample being analysed (sample cell) and the other beam; the reference beam, passes through a cuvette containing a solvent (reference cell). The intensities of the beams are measured by electronic detectors and a comparison of the two is made. The light that is passed through the reference sample should have suffered very little or no light absorption and is defined as I0 and the intensity of sample beam is defined as I.

Figure . Schematic of UV/Vis Spectrometer

The spectrometer automatically scans all the constituent wavelengths; the near UV (185 – 400 nm), visible (400 – 700 nm) and very near infrared (700 – 1100 nm). Most commercial spectrophotometers cover a spectral region of 185 – 900 nm (Rouessac & Rouessac, 2007).

If no light is absorbed by the sample compound then I = I0, but as the sample absorbs light resulting in I < I0, then this difference is plotted on a graph of absorbance verses wavelength (λ) as in figure 2 below. This absorption can be transmittance (I/I0) or absorbance (log I0/I). The wavelength of the maximum absorbance is defined as λmax. The most common solvents used in UV/Vis spectroscopy are water, ethanol, hexane and cyclohexane. It is best to avoid solvents that have a double or triple bond in their molecular structure such as bromide or iodine as the absorbance of the sample will be proportional to the molar concentration of the sample cuvette.

Synthesis metal nanoparticle

Figure . UV–Vis spectrograph of copper nanoparticles (Free Patents Online, 2012)

UV/Vis spectroscopy can be used to identify copper nanoparticles as they have absorption bands at 250 nm (Cu+), 320–370 nm and 400–440 nm (charge transfer bands of O–Cu–O and Cu–O–Cu complexes), 520–580 nm (copper nanoparticle plasmon resonance) and 620–850 nm (d–d transitions in Cu2+ions). (Pestryakov, et al., 2004). 

1.5.2 Atomic Absorption Spectroscopy

Atomic absorption spectroscopy (AAS) is a sensitive analytical tool for the determination of more than 60 elements. AAS measures the concentration of gas-phase atoms by analysing the absorption of light. Analytical methods using atomic absorption are very likely to be highly specific, the reason being that the atomic absorption lines are extremely narrow and because electronic transition energies are unique for each different element (Skoog & West, 1980). The analyte atoms or ions must be vaporised in a flame or graphite furnace. The atoms will absorb the UV or visible light and move to higher electronic energy levels. The amount of absorption then determines the concentration of the analyte. The concentration measurement is usually determined using a standard/working curve after the instrument has been calibrated using known standards.

Figure . Schematic of AAS Spectrometer

The light source is typically a hollow cathode lamp consisting of the element being measured, which consists of a tungsten anode and a cylindrical cathode sealed in a glass tube that is filled with argon or neon gas at pressures of 1 - 5 torr. The cathode consists of the metal of the desired spectrum or it can serve as a support layer of the desired metal. A variety of hollow cathode tubes are available commercially, some of which consist of several metals, so they permit the analysis of more than one element (Skoog et al. 2004). Lasers can also be employed as they are intensive enough to excite the atoms to the higher energy levels so they allow atomic absorption and atomic fluorescence. The downside of these narrow band light sources is that this only allows for the measurement of one element at a time (Tissue, 2000).

For light separation and detection, atomic absorption spectrometers use monochromators/diffraction gratings and detectors for UV/Visible light. The main purpose of the monochromator is to isolate the absorption line from the background light due to interferences. A bandpass interference filter can be used to replace the monochromator. The most common detector used in AAS is a photomultiplier tube (Tissue, 2000).

1.5.3 Stripping Voltammetry (SV)

Stripping voltammetry (SV) is now a significant trace analytical method, particularly used in the determination of metals in the environment (Skoog et al., 2004). There are two very sensitive methods; anodic and cathodic stripping voltammetry, which are used to measure traces of metal, and thus can be used when analysing copper nanoparticles. They are carried out in two stages: preconcentration and re-dissolving. The analyte is first deposited into a small volume of mercury, usually from a stirred solution.

Figure . Stripping Voltammetry; the effect of electroplating and re-dissolving on the graph (adapted from Foo, 2010)

In preconcentration a small fraction of the target electroactive analyte is deposited on an electrode which is kept at a constant potential and submerged in a stirred solution of the sample. The electrode can be a hanging mercury drop, a cylinder of vitreous carbon that is covered by a film of mercury, or a rotating carbon disc with mercury salt added to the solution. In each of these cases the surface of the electrode gives a combination of M(Hg) with Mn+ metallic analytes (Figure 4).

In re-dissolving (also called the voltammetric step) after the electro-deposition, the mixing of the solution is stopped and the potential difference is gradually reduced between the electrodes. Due to the reversible redox reactions and an electrochemical oxidation, the analyte is re-dissolved. The elements are then identified by their oxidation potential (Pestryakov et al., 2004).

In anodic stripping, during the deposition step, the working electrode behaves as a cathode and during the stripping step it behaves as an anode, with the analyte being oxidized back to its original form. In a cathodic stripping method, during the deposition step the electrode behaves as an anode and as a cathode during stripping. As the material is deposited into a much smaller volume than the bulk solution volume, the analyte can be concentrated by factors of 100 to more than 1000 in the deposition step (Skoog et al., 2004).

Many other variations of the stripping technique have been expanded from the hanging drop and vitreous carbon methods. For example, different cations have been determined by electro-deposition on a platinum cathode. The amount of electricity necessary to remove the deposit is then measured using colorimetry. This method is also very advantageous for trace analyses.

1.5.4 Fourier Transform Infra Red (FTIR)

Fourier Transform Infrared Spectroscopy (FTIR) is a valuable tool for identifying either organic or inorganic chemicals. It can be employed to estimate or determine precisely the number, degree, or amount of some of the components of an unknown mixture. FTIR can be used in the analysis of gasses, liquids and solids. The term FTIR refers to a quite recent development in the approach in which the data is collected and then converted to a spectrum from an interference pattern. Modern FTIR instruments are much faster and quite a bit more sensitive than the older dispersive instruments, as they are computerised (Exova, 2012). 

FTIR instruments detect all wavelengths and these can be detected and measured simultaneously as they contain no dispersing element. An interferometer is used in place of a monochromator and is used to construct interference patterns containing the infrared spectral information. The same kinds of sources are used in FTIR spectrometers that are used in dispersive instruments. The majority of benchtop FTIR spectrometers use a single-beam.

In order to acquire the spectrum of a sample, first the background spectrum is obtained by Fourier transformation of the interferogram from the background i.e. CO2, solvent and water. The next step is obtaining the spectrum of the sample. And the last step is calculating the ratio of the background spectrum to that of the single beam sample spectrum and a graph is plotted with absorbance or transmittance versus wavelength or wavenumber (Skoog et al., 2004)

Figure . Schematic of FTIR

FTIR can be used in the identification of chemicals from drugs, coatings, contaminants, paints, polymers, etc. FTIR is possibly the foremost tool for the identification of chemical bonds i.e. functional groups. The chemical bonds can be identified from interpretation of the infrared absorption spectrum and the wavelength of light absorbed.

The FTIR spectra obtained from pure compounds are like a molecular "fingerprint" as they are quite unique. Organic compounds on the other hand have very detailed, rich spectra while inorganic compounds are usually much less complicated. For the majority of common materials, the spectrum of an unknown compound can be identified when compared to a library of known compounds.

FTIR can be used for quantitative analyses as the absorption strength is proportional to the concentration. Usually these are somewhat simple forms of tests in the concentration range of just a few ppm up to the percent level (Exova, 2012). 

1.6 Instrumentation

In this section I will describe the instrumentation used for the detection of the model drug, analgesic.

1.6.1 Raman Spectroscopy

Most molecular vibrations energy corresponds to that of the electromagnetic spectrum or the infrared region. Molecular vibrations can be detected and measured indirectly in a Raman Spectrum (Williams & Fleming, 2008). Raman spectra are achieved by irradiating a sample with a strong source of visible monochromatic radiation and the quantity of material that can be measured is now in the order of a few milligrams. A mercury arc was employed in early investigations, now this source has been superseded by the use of high-intensity gas or solid lasers (Skoog & West, 1980). Raman spectroscopy is concerned with the radiation scattering from a sample, and this scattered light is examined through a spectrometer using photoelectric detection. This scattering takes place when an incident photon interacts with the electric dipole of a molecule. This scattering can be elastic or inelastic. Light can be considered as an electromagnetic wave. As the beam of monochromatic exciting radiation passes through the molecules of a sample, the molecules can undergo a change in molecular polarisability as they vibrate. The electron cloud around the molecule begins to contract and elongate bringing about a change to the polarisability. This will cause a modulation in the scattered light at the vibrational frequency and the induces oscillating dipole radiates not only at the frequency of the incident light but in addition at frequencies corresponding to the sum and the difference of this frequency and the molecular vibrational frequencies (Willard et al., 1974)

Most of the incident photons are elastically scattered by the molecule, this is referred to as Rayleigh scattering. In this Rayleigh scattering the energy of the incident photons is equal to the energy of the photons that are scattered. A small fraction of the light is scattered at energies that are different than that of the incident photons, this occurs in approximately one in every million collisions. This is inelastic scattering and is called the Raman effect and was first observed in 1928 by Chandrasekhara Venkata Raman who was awarded the Nobel Prize in 1930 for his work.

Raman spectroscopy relies on the inelastic scattering (also called Raman scattering) of monochromatic light, usually in the form of a laser which, when shone onto a sample, will become scattered by the molecule in the sample. This involves a quantised exchange of energy between the scatterer and the incident photon that results in weak scattered lines that are separated from the exciting line by frequencies equal to that of the vibrational frequencies. Raman follows equation 1:

Equation

Where:

is the frequency of the incident photon

is the initial energy

is the frequency of the scattered photon

is the final energy state of the molecule

When the incident lights energy is not strong enough to excite the molecules from the ground state to the lowest electronic state, the molecule will be excited to a virtual state in between the ground and lowest electronic state. As the electron cannot stay long in the virtual state it will go back to the ground state.

Figure . Schematic diagram of Rayleigh, Stokes and Anti-Stokes Raman Scattering

If the electron returns to the position it originated from, then the wavelength of the scattered light is equal to the wavelength of the light source, this is called Rayleigh scattering. The electron can also go to a vibrational state that is different from where it is excited; this results in an energy difference between the emitted photon and the incident photon. If the emitted energy is smaller than the incident energy, the process is called the Stokes scattering. If the emitted energy is larger than the incident energy then this is called the anti-Stokes scattering.

Surface Enhanced Raman Spectroscopy (SERS)

Conventional Raman spectroscopy has the disadvantage of low signal strength in comparison with other forms of optical spectroscopy such as fluorescence. On its own it is not ideal for investigating molecular conformation in solution at low concentrations, which is a common requirement in the life sciences. Surface Enhanced Raman Spectroscopy (SERS) is a surface-sensitive spectroscopic technique that brings together the technologies of modern laser spectroscopy and the interesting optical properties when molecules are connected to nanometre-sized gold, silver and copper structures, resulting in impressively increased Raman signals. It provides spectroscopic information with low background. It can increase the Raman signal considerably, which means the technique can detect single molecules, making SERS an ideal tool for future bio-physical investigation (University of Exeter, 2012).

Figure . Schematic diagram of SERS on a roughened metal surface (adapted from Semrock, 2012) and using copper nanoparticles (right)

Molecules absorbed onto specific metallic substrates or in close proximity of metal nanoparticles; typically gold, silver and copper, exhibit this enhancement in the Raman signal. Enhancements are reported to be as high as 1014. Chemical and electromagnetic field enhancement of vibrational modes bring about this increase in signal. It is not altogether clear if this enhancement mechanism has any effect on the information obtained from Raman spectra.

There are many reports using colloidal silver and gold with SERS and they are ideal substrates for making measurements in a solution. When a solution of the sample to be analysed, such as analgesics, is mixed with a colloid solution of nanoparticles, the molecules are absorbed onto the surface of the nanoparticles in the colloid. The molecules obtain molecular resonance due to the close proximity and the result is enhancement of the signal by interaction with the plasmons on the metal surface. This combined enhancement creates a Raman signal with sensitivity close to that of fluorescence and provides the structural information of Raman spectroscopy together with extremely sensitive detection limits.

1.7 Conclusion

The use of low cost, reliable chromatographic and spectrographic techniques in the detection of analgesics and other drugs are advancing steadily and the need for this detection is a very real necessity. Surface enhanced Raman spectroscopy is a very sensitive spectroscopic method with a single molecule detection limit. It has a good multiplexing capability, so much so that a Raman signal is enhanced up to 1014 fold. It provides good spectroscopic information with low background. Even though it is still in its infancy as there is much advancement to the technique regularly, it is receiving careful attention due to its extraordinary properties. The use of SERS is becoming more common, but using silver and gold which are routinely used in SERS as the roughened metal surface or nanoparticle, can be quite costly. The use of copper is an ideal low cost, easily synthesised, readily available alternative. Copper nanoparticles are easily synthesised and using different methods, and their size and structure can be easily determined using analytical tools such as AAS, UV/Vis spectrometry, FTIR and many more. And this low cost substrate will help the advancement of new techniques and variations to SERS in the future.