|Year : 2019 | Volume
| Issue : 2 | Page : 37-41
Surface-Enhanced Raman Spectroscopy to study the biological activity of anticancer agent
Guoyu Qiu1, Xiaohui Xu1, Lupeng Ji2, Ruiping Ma3, Zilong Dang4, Huan Yang5
1 Department of Traditional Chinese Medicine and Chemical Drug, Lanzhou Institutes for Food and Drug Control, Lanzhou, Gansu, China
2 Department of Medicine, The Fifth People's Hospital of Zhuhai, Zhuhai, Guangdong, China
3 Department of Food, Gansu Province Product Quality Supervision and Inspection Research Institute, Lanzhou, Gansu, China
4 Department of Pharmacy, First Hospital of Lanzhou University, Lanzhou, Gansu, China
5 Shanghai Cell Therapy Engineering Research Center, Jiading, Shanghai, China
|Date of Submission||10-Apr-2019|
|Date of Acceptance||23-Jun-2019|
|Date of Web Publication||28-Jun-2019|
Dr. Huan Yang
Shanghai Cell Therapy Engineering Research Center, No. 1585, Yuan Guo Road, Jiading District, Shanghai 201805
Source of Support: None, Conflict of Interest: None
Surface-Enhanced Raman Spectroscopy (SERS) is a sensitive and selective spectroscopic technique for the detection and characterization of analytes, which are adsorbed on suitable metal surfaces. SERS as a strategy has been widely used in detecting target molecules, and the screening of drug activity is mainly to study the biological effects of drug combined with target, so a study on biological activity of anticancer drug based on SERS is a concern to some researchers. SERS combines the advantages of positive identification of molecules in situ, stand-alone detection, well-established instrumentation with high sensitivity, and its noninvasive detection. In this paper, we review on the application of SERS in studying anticancer agent from three aspects, such as studying the effects of anticancer agent on cancer cells, detection of anticancer agent in human plasma, and testing the interaction between anticancer agent and DNA or protein.
Keywords: Anticancer agent, biological activity, substrate, surface-enhanced Raman scattering
|How to cite this article:|
Qiu G, Xu X, Ji L, Ma R, Dang Z, Yang H. Surface-Enhanced Raman Spectroscopy to study the biological activity of anticancer agent. Cancer Transl Med 2019;5:37-41
|How to cite this URL:|
Qiu G, Xu X, Ji L, Ma R, Dang Z, Yang H. Surface-Enhanced Raman Spectroscopy to study the biological activity of anticancer agent. Cancer Transl Med [serial online] 2019 [cited 2020 Feb 24];5:37-41. Available from: http://www.cancertm.com/text.asp?2019/5/2/37/261827
| Introduction|| |
Cancer is a kind of disease that possesses the potential to invade or spread to other parts of the body due to abnormal cell growth. Cancer is the second leading cause of death globally and is responsible for an estimated 9.6 million deaths in 2018 globally, and about 1 in 6 deaths is due to cancer. Anticancer drugs are the weapon to fight against cancer. Currently, about 130–150 anticancer drugs have been approved for sale in various countries., In the process of drug research and development, the anticancer drug fails due to unsuitable drug targets, unsuitable molecules, equivocal conclusions, and unmatched patients. In recent years, Raman spectroscopy is being used to screen anticancer drugs. Raman scattering was first discovered by Raman in 1928 in liquids as a new radiation from the vibrations of molecules and was also reported by Landsberg and Mandelstam in crystals independently almost at the same time. It describes the interaction between light and the vibrations of molecules or atoms in a solid. The active pharmaceutical ingredients contain aromatic or conjugated domains with strong Raman scattering activity. Hence, Raman spectroscopy is an attractive alternative conventional analytical method for pharmaceuticals, which contains information on molecular vibrations and provides a highly specific fingerprint of the target molecule. Raman spectroscopy has been widely used in cell imaging andin vivo imaging due to the advantages of narrow spectra and a near-infrared excitation scheme. However, direct applications of Raman spectroscopy are severely restricted by its low signal intensities which may be overcome by increasing the intensity of Raman scattering, either by resonant excitation or by surface enhancing, as shown in [Figure 1]. Recent research efforts have been focused on the enhancement of weak Raman signals, surface-enhanced Raman spectroscopy (SERS). SERS, a vibrational spectroscopic technique,,,,,,,, as a strategy has been widely used in the field of analysis. SERS technology has a unique advantage of nonlabeled nondestructive detection with high sensitivity at a molecular level, which is suitable for life science research. The sample can be observed on the water-based or physiological salt water basis, and the characteristic spectral line of water based is the reference for absolute strength. Fluorescence quenching can be achieved by detecting the energy transfer between molecules and the substrate, so as to avoid the interference of spontaneous fluorescence or impurity fluorescence of biological samples and obtain the clear Raman spectrum of fluorescent substances. The adoption of SERS based on probes and appropriate light guide system makes it possible to achieve real-time and highly sensitive detection on the body surface and internal tissues, which provides a new method for biomedical research. SERS has been the de facto verification technique for screening anticancer agents rapidly, with high sensitivity, selectivity, and specificity to the identification of interactions between the anticancer agent and the target. The relationship between SERS, SERS substrates, and anticancer drug is shown in [Figure 2].
|Figure 2: The relationship among SERS, SERS substrates, and anticancer agent|
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| The Substrate of SERS|| |
In the basic process of SERS, the analyte is adsorbed onto a roughened metal surface of a suitable metal, usually silver or gold. On excitation of this surface with a laser beam, change in polarizability of the analyte occurs in a direction perpendicular to the surface, leading to enhanced scattering. Active substrate is the key for SERS to be used in screening anticancer agent. The strength of SERS effect comes from the material used in its substrates, also influenced by the size and shape of the substrates. The mechanisms of SERS include physical and chemical enhancement mechanisms. Physical enhancement mechanism mainly describes how the localized surface plasmon resonance on the surface of the noble metals and transition metals could enhance the electromagnetic field.,, Chemical enhancement mechanism describes the interaction between the compounds and enhancement substrate, which can be classified as the chemical bonding, surface complex, or photo-induced charge transfer. The shape, morphology, space, assembly, and distance of the substrates may affect the SERS activity. Therefore, the structures of the substrate are very important for a particular metal. Sharp corner of the metals provides better enhancement for SERS detection. Recently, researchers have developed many kinds of SERS substrates with various structures. They are the basic parts of SERS substrate structures, especially when the SERS substrates facilitate other functions of the analytes. Most important substrates are metal oxides, graphene, silica, and polymers. The applied metal oxides mainly include CuO, ZnO, TiO2, and Al2O3. Prepared metal oxides are stable under various conditions, which is the advantage of metal oxides. Graphene is to build Ag/ZnO/reduced graphene oxide (rGO) nanocomposite or prepare rGO/AgNPs as a SERS substrate. The third substrate is silica, and its advantage is to prevent aggregation by coating the SERS-active points and separating them to obtain much better SERS effect, which can also improve the SERS stability of the substrates. Polymer substrate is another important kind which could be conveniently and efficiently built by electrospinning technique. Among these substrates, Ag, Au, and Cu have the high SERS activity when they work together as a substrate. In general, Au and Ag are most often used as SERS substrates because they are air stable, and Cu is more reactive. All three metals have presented most of the visible and near-infrared wavelength ranges, where most Raman measurements occur and hence are frequently used [Figure 3].
|Figure 3: Wavelength ranges where Ag, Au, and Cu have been well-characterized and are established to support SERS|
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| The Application of SERS in Studying Anticancer Agent|| |
Effects of anticancer agents on cancer cells
At present, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) is the most widely used assay to screen a large number of anticancer agents for their cytotoxic effects on various cancer cells. MTT is a yellow, water-soluble reagent where metabolically active cells reduce the tetrazolium salt into colored formazan product. It has also been reported that MTT assay coupled to surface-enhanced resonance Raman scattering (SERRS) spectroscopy could be one of the best approaches to study the effects of anticancer agent toward cancer cells. Since it is a challenge to avoid autofluorescence and minimize light absorption from tissue, one way to amplify weak Raman signals is to employ SERS using sharp ending metal surfaces, such as nanostars. As a result of utilizing different shape plasmonic-enhanced Raman signal from anticancer agents, it has been possible to obtain a real-time cellular imaging of cancer cells. SERRS has been used to develop and optimize a novel and quantitative MTT assay for living cancer cell viability. This highly sensitive method depends on two factors for formazan signal enhancing: the addition of Au nanoparticles and the resonance effect from excitation at 632.8 nm. Results show that the background elements, such as excessive MTT residues, serum, and the agent, did not interfere with detection of formazan. Moreover, the detection limit of formazan is as low as 1 ng/mL. With the use of this method to quantify metabolically viable cells, dose–response curves of treated and untreated cells with the agent were constructed. The results also show that the Raman signal generated is dependent on the degree of activation of cells. In comparison to the traditional method, advantages of this method are its rapidity, high selectivity, high precision, and cost-effectiveness without time-consuming steps and any modifying or labeling procedure. MTT assay coupled to SERRS may help researchers screen anticancer agent by administering on cancer cells, and this SERRS-based MTT assay is shown in [Figure 4]. In addition, SERS was also used to study cancer cells' morphology or the change in biomarker caused by anticancer agent. Some researchers have used gold nanostars to enhance plasmonic-enhanced Raman signal from an anticancer drug conjugated to gold nanostars in imaging of cancer cells.,, For example, cetuximab is the first monoclonal antibody drug that targets epidermal growth factor receptor (EGFR) which is overexpressed in cancer cells. It easily binds to EGFR, thereby downregulating the receptor, blocking EGFR-mediated tyrosine kinase activity, and inhibiting cellular proliferation. Thus, EGFR–cetuximab binding can be quantified to monitor receptor status and the prognosis of cancer therapy. It was reported that Chung et al. had used SERS imaging based on silica-encapsulated gold nanotags to assess the inhibitory effect of cetuximab on EGFR expressed on cancer cells. However, intense Raman signals were also obtained in detecting living cancer cells in the absence of metal (such as Au or Ag) as Raman enhancers, and a series of novel Raman beads with Raman signals in the biological Raman-silent region have been developed, which was a new strategy to image organic polymeric nanoparticles free from the use of metals as substrate.
|Figure 4: The procedure of the surface-enhanced resonance Raman scattering-based 3-(4,5-dimethyl--2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay|
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Detection of anticancer agent in human plasma
It is essential to detect anticancer agent in human plasma as we need to study pharmacokinetics and pharmacodynamics of agent. So far, liquid chromatography–tandem mass spectrometry (LC-MS/MS) is applied widely in this field. However, LC-MS/MS is complex and demanding. Recently, a new method for quantitation of anticancer agent in human plasma based on SERS and multivariate calibration using partial least squares regression (PLSR) has been proposed. For example, an anticancer drug, imatinib, is used as the object of research based on SERS by the researchers. The purpose is to improve the treatment response and minimize the risk of adverse reactions in chronic myelogenous leukemia and gastrointestinal stromal tumor patients. The best PLSR model is obtained with three latent variables in the range from 123 to 5000 ng/mL of imatinib, providing a standard error of prediction of 510 ng/mL. This method is validated in accordance with international guidelines, through the estimate of figures of merit, such as precision, accuracy, systematic error, analytical sensitivity, detect limitation, and quantitation. Moreover, the feasibility and clinical utility of this approach have also been verified using real plasma samples taken from de-identified patients. The results are in good agreement with a clinically validated LC-MS/MS method. The new SERS method showed simplicity, short analysis time, and good sensitivity and could be considered as a promising method for detecting anticancer agent in human plasma. It was reported that the anticancer drug, paclitaxel, was detected and quantified in blood plasma using microwave-treated gold film-polystyrene beads with modified gold surface topography as SERS substrate. SERS has also been applied for therapeutic drug monitoring (TDM), monitoring real-time physiological load of therapeutic drugs most commonly in plasma or serum of patients at a known time related to administration in terms of relevant clinical parameters such as target range and pharmacokinetics of the drug. TDM application of SERS for flucytosine monitoring in a blood sample using microporous membranes in the vertical flow assay to separate flucytosine from whole blood sample is shown in [Figure 5].
|Figure 5: Schematic of the passive vertical flow assay setup and interface with paper SERS sensors for easy Raman interrogation|
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Interactions between anticancer agent and DNA or protein
A study on a number of drugs and their interactions with DNA has been reported, including anthracycline antitumor antibiotics, daunorubicin, and doxorubicin (DOX) using SERS with silver colloid plasmonic enhancer. It is well known that many anticancer drugs are redox active, which is very important and critical to their biological function. For instance, anthracyclines are a class of anthraquinone-derived drugs used in anticancer therapy; they inhibit DNA and RNA synthesis after intercalation between the oligonucleotide bases. It has been reported that the one-electron reduction of quinone anticancer drugs is more damaging at a biological level because of the possibility of associated generation of reactive oxygen species., It is important to improve our understanding on the role of these interactions in the therapeutic action of anticancer drugs. It is, therefore, important to be able to study how the properties of an electroactive intercalator change with its binding to DNA as a consequence of changes in the redox state of the intercalator itself. Perspective analytical applications of novel SERS nanostructures as a method to study anticancer agent interactions with DNA have recently been reported. The work focused on the development of a laboratory nanobiosensor for screening anticancer agent. Anticancer agent interactions with DNA bound to the nanostructured SERS were assessed. Particular attention to study new anticancer agents has been paid to increase the agent efficacy toward killing cancer cells while limiting their devastating effects on healthy cells through the targeted approach based on cancer cell biorecognition strategy. In addition, anticancer agents damage cancer cells due to their strong interaction with DNA. SERS spectroscopy is used to observe the drug–DNA reactivity. For example, the self-assembled monolayer protected gold-disk electrode (AuDE) was coated with a rGO and decorated with plasmonic gold-coated Fe2Ni@Au magnetic nanoparticles functionalized with double-stranded DNA (dsDNA, a sequence of the breast cancer gene BRCA1). The nanobiosensors AuDE/SAM/rGO/Fe2Ni@Au/dsDNA were then subjected to the action of a model anticancer agent, DOX, to assess the DNA modification and its dose dependence. The SERS measurements have corroborated the DOX intercalation with DNA duplex, whereas the electrochemical scans have indicated that the DNA modification by DOX proceeds in a concentration-dependent manner. The new biosensor is sensitive to anticancer agent interaction with DNA. The proposed nanobiosensors can be applied in the first stage of the anticancer agent development for testing the interactions of anticancer agent with DNA before the agent efficacy can be assessedin vitro and in vivo. [Figure 6] shows a typical set of SERS spectra of mitoxantrone collected in the course of an electrochemical SERS experiment scanning the potential between −0.6 and −1.2 V. However, SERS assays for the detection of unlabeled target DNA have some drawbacks including requirement for separation steps using magnetic beads. Separation steps are labor intensive, expensive, and increase the risk of sample contamination. Now, Lierop et al. have presented an increased SERS response upon hybridization of target DNA without the need for additional separation steps and is fully compatible with polymerase chain reaction. In addition, SERS is used to detect the changes in related enzymes when agent binds to the target. Variation in enzyme activity due to agent was measured from changes in intensity of a characteristic peak of the SERS spectrum that was directly correlated with the concentration of the effect of agent.
|Figure 6: SERS spectra of mitoxantrone bound to dsDNA at increasing negative potentials|
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| Conclusion|| |
SERS has gained remarkable results in both theory and application in screening of anticancer drug. Researches based on SERS are underway, not only Raman scattering will be improved, but also the second harmonic, fluorescence, and light absorption on the surface of SERS activity will be enhanced or quenched. There are some disadvantages to SERS such as it is hard to control and lack of other biocompatible substrates. In addition, SERS requires the presence of chemical groups such as aromatic ring, heterocyclic ring, amino group, carboxylic acid group, phosphorus, sulfur atom, and other groups with Raman activity which limits the detection of objects to some extent. SERS technology still needs to make great progress in finding suitable Raman label, improving its stability, easy operation, and expanding the application scope of analysis. With further understanding of SERS principle, the stability of laser and the improvement of detector sensitivity, as well as the progress of weak signal extraction and processing technology, will be improved. Hence, SERS is very promising in screening anticancer drugs in the future.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
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