Probing the component distribution and the corresponding location mapping of biological macromolecules or biopolymers is one of the fascinating research fields because it is crucial to inquire and understand the novel properties of vital materials in the living body, even in the remediation of tissues and the therapy of many diseases [1
]. For example, Nucleic acids, a category of biological macromolecular compounds, can be synthesized through polymerizing nucleotides with different stoichiometric coefficients and different arrangements. They play important roles in storing and transmitting genetic information during numerous biological processes including replication and synthesis of proteins, heredity and variation of life phenomena, and so on. Therefore, detecting nucleotide sequences and mapping their corresponding locations are of great importance in life science and biomedical technology. Nowadays, there are many methods including the Maxam-Gilbert sequencing and Chain-termination techniques which have been developed to determine DNA sequences and investigate the structure and function of nucleic acids [5
]. Especially, the configuration, rotation period, major groove and minor groove of DNA molecule were firstly probed at the atomic level by scanning tunneling microscopy (STM) in 1989. It provided a visual and reliable demonstration of the double-helix of DNA molecules [8
]. However, detecting the molecular characterization and its correlative structural information is still a challenge in the last decades.
Except for DNA and RNA, proteins are another kind of biological macromolecules which are consisted of one or more long chains of amino acid residues. Each kind of protein molecule has a unique three-dimensional structure and carries out diversified biological functions in the living body. According to the difference of amino acid sequences, proteins can also be divided into several categories such as Antibody, Enzyme, Messenger, Transport and Storage etc. [9
]. Undoubtedly, discovering simultaneously the tertiary structure and corresponding amino acid sequence of a protein can provide important clues to how a protein performs its function [10
]. Even more, these related investigations will not only contribute to the understanding of vital biological processes, but also be beneficial to the design of new ligands applied to the development of new drug molecules. However, many traditional analysis techniques (scanning electron microscopy, scanning probe microscopy, X-ray diffraction, nuclear magnetic resonance, etc.) usually can only provide the structural or chemical information [12
]. Therefore, a tool which can combine topography with chemical information will accelerate the research of proteins.
Tip enhanced Raman spectroscopy (TERS), a newly-developed technique combining scanning probe microscopy (SPM) with the Raman spectroscopy together, can provide chemical analysis and structural topography of a sample with a high spatial resolution [16
]. In TERS experiment, the Raman signal of molecules over the sample surface is enhanced due to the coefficient of the surface plasmonic resonance and the lighting-rod effect. When a probe scans over the sample surface at a certain speed, the topography at a resolution better than 20 nm and the label-free Raman mapping can be obtained simultaneously. Besides, atomic force microscopy based TERS (AFM-TERS) inherit two advantages from AFM. Firstly, AFM can be operated in liquid conditions, which makes it possible to study biological samples in native environments [18
]. Secondly, AFM may be used in two different operating modes, namely the contact mode and the tapping mode. When the fragile samples, such as RNAs [21
] and lipid bilayers [22
], are investigated, the tapping mode can be chosen to avoid the damage to the sample since the force between the tip and the sample is smaller in this mode. Due to these reasons, AFM-TERS is gradually employed to investigate masses of biological samples, such as nucleic acids [23
], proteins [24
], pathogens [26
], lipids and cell membranes [27
In this review, the working principle of the TERS setup will be presented and discussed. Then the applications of AFM-TERS in nucleic acids, proteins and pathogens will be presented. All of these results indicate that the simultaneous acquisition of the structural and chemical information at the same scanning point of a sample through AFM-TERS technique can not only help to understand the relationship between the structure, composition and function of a biomolecule, but also visually clarify the variation mechanism of lives in different physiological or pathological conditions, thus giving an important guidance to future pharmaceutical sciences.
3. Applications of AFM-TERS in Biology
How to demonstrate the relationship between the structure, function and chemical information of biological molecules in natural environments has always been one of the big challenges in life science. AFM-TERS can provide both the structure and chemical information of a sample at the nanoscale simultaneously in various experimental conditions, which causes AFM-TERS to be widely used in biological samples such as nucleic acids, proteins, pathogens, lipids and cell membranes, etc.
3.1. Nucleic Acids
TERS technique has been widely applied to characterize nucleic acid bases due to its characteristic of small sample amount, high sensitivity and direct-sequencing [19
]. Five nucleic acid bases including adenine (A), cytosine(C), thymine (T), guanine (G), and uracil (U) were involved in such analysis. The earlier TERS investigations were focused on the nucleic acid nanocrystals [85
]. For example, Regina Treffer et al. demonstrated the reproducibility of AFM-TERS by investigating the single-stranded adenine and uracil homopolymers on the different kinds of substrates. Later, the same equipment was used to study single-stranded calf thymus DNA with arbitrary sequence [89
]. The result proved that it was possible to distinguish different nucleobases clearly. The combination of sensitivity and reproducibility shows that TERS can meet the crucial demands for a sequencing procedure. Subsequently, a series of successful TERS investigations of single-stranded RNA, single-stranded DNA and double-stranded DNA have come forth.
E. Bailo and V. Deckert presented a TERS experiment for sequencing a single-stranded RNA of cytosine(poly(C)) in nanomolecular quantities [44
]. With an Ag-covered AFM tip, the topography of a single-strand RNA was imaged (Figure 4
a) and then eight Raman spectra (including seven from different positions along the RNA chain and one from the reference) were measured by TERS (Figure 4
b). According to the article, the signal-to-noise ratio (SNR) of the TERS spectrum was obtained by dividing the intensity of the most intense Raman band by two-times the standard deviation of the noise level measured in a signal-free section of the spectrum. The SNR here was about 200. Every TERS spectrum exhibited 30–60 bases beneath the tip (about 20 nm) and each base contributed a SNR about 3–7 to the spectrum. This indicated that the visualized image with high contrast for the direct-sequencing of nucleic acid became feasible.
Najjar et al. presented another TERS experiment to investigate combed double-stranded DNA bundles [90
]. With a tip covered by a Ag/Au bilayer, all of the DNA nucleobases and those backbones containing phosphodiester and sugar groups could be identified by the Raman signal of TERS. This indicated that DNA hybridization could be detected via the TERS technique.
Lipiec et al. characterized the molecular structure of double strand breaks (DSBs) by the TERS technique [23
]. The controlled and irradiated samples were firstly imaged by the AFM technique, which showed that the plasmids went into a relaxed state or a linear shape instead of their supercoiled quaternary structure following ultraviolet C (UVC) exposure. Then, potential cleavage sites were found according to the TERS spectra. Comparing these images, it could be concluded that the DSBs mainly resulted from the 3′- and 5′-bonds of deoxyribose upon UVC-exposure and the 3′-bond was the most likely type of DSBs since it accounted for more than half of the breaks detected.
The examples above show that AFM-TERS has succeeded in distinguishing the respective nucleobases, detecting DNA hybridization and characterizing the molecular structure of DSBs. AFM-TERS permits chemical identification at a scale of 10 nm (which is smaller than the size of the tip apex radius). It means there are 30–60 bases responsible for the Raman signal in every spectrum. The resolution is not enough to unambiguously distinguish a single base. It’s well known that the resolution of AFM increases with the decrease in the size of the scanning tip apex. However, for AFM-TERS, decreasing the size of apex means a thinner metal layer on AFM tip. As a result, the tip might lose its TERS activity. In order to increase the resolution of AFM-TERS, metal nanowire attached to a tip is another alternative in AFM-TERS. However, the related work has not been published yet. Recently, Rui Zhang et al. reported the outstanding capability of STM-TERS to distinguish individual DNA bases (adenine and thymine) with a super-high spatial resolution of ~0.9 nm [57
]. This work opened a new way to achieve single-molecule DNA sequencing on surfaces by TERS technique. It should be noted that the experiments were carried out under the condition of low temperature and high vacuum. Obviously STM-TERS has a higher resolution than AFM-TERS not only because of the massive metal probes, but because of the extreme conditions. However, it is impossible for the biological samples to keep alive under such extreme conditions.
Proteins are the executors of physiological functions and the direct embodiments of life phenomena. The study of the protein structure and function will visually clarify the variation mechanisms of lives in different physiological or pathological conditions.
The heme protein cytochrome c (Cc), consisting of heme, covalently bound to the protein through thioether links and a Fe-amino coordination, is one of the proteins which was investigated by TERS in the early stage. In this specific experiment, Raman properties of Cc were detected by AFM-TERS and SERS at the same time [91
]. The result showed that with an Ag coated AFM-tip, TERS spectra could present the Raman signal of both the heme and amino acids Cc. However, the information of amino acids could not be observed in SERS. This proved that with superior spatial resolution and stronger enhancements, TERS could give more structural information about large biomolecules compared with SERS.
Additionally, a series of contrast experiments about bovine serum albumin (BSA), phenylalanine and tyrosine were carried out. In the experiments, Raman signals originating from conventional Raman, SERS and TERS at the same excitation wavelength were compared [92
]. It was found that the relative spectral intensities of the Raman peaks were usually different, but the positions of them were unchanged in all these three methods. These research results demonstrated that TERS characterization could not only yield spectroscopic fingerprints similar to conventional Raman and SERS, but provide a higher spatial resolution at nanometer scale. The information of TERS would provide more useful guidelines for understanding the structures and functions of biological molecules.
With these incomparable advantages, more and more complex protein structures were analyzed by TERS technique. For example, amyloid fibrils, caused by protein aggregation inside the human body, have been acknowledged to be responsible for many neurodegenerative diseases, such as Alzheimer’s disease, Huntington’s disease and prion diseases. In the early years, it could not be fully understood what caused the amyloid-like protein aggregations. Recent researches on topography and chemical characterization detected by TERS offered much useful information [93
]. One of the typical TERS experiments related with amyloid fibers is about insulin protofilaments, fibril polymorphs and their topographies. In the experiment, the surfaces of two insulin fibril polymorphs with flat and twisted morphologies were investigated. TERS spectra showed that (see Figure 5
] both twisted fibrils and flat fibrils had different types of amino acid residues on their surfaces. Cys was potentially involved in the form of the twisted fibrils, but it was most likely nothing to do with the propagation of the flat fibrils. These results demonstrated that there were different formation mechanisms for twisted fibrils and flat fibrils.
These above results present the plausible use of TERS technique to characterize surface amino acid components and protein secondary structures, which are helpful to understand the mechanisms of fibril growth and propagation. Therefore, TERS characterization can be widely used to define the keys to preventing or slowing down the progression of various neurodegenerative diseases.
Successfully detecting the structure, dynamics and function of pathogens is crucial for public health since these works can provide some guidelines on the prevention, diagnosis and therapy of the diseases.
The first kind of pathogens detected by AFM-TERS was Gram-positive bacteria Staphylococcus epidermis [43
]. With the AFM-TERS technique, the topography of a single epidermis cell and AFM-TERS spectra detected at the different positions of the bacterial surface were investigated. The results indicated that most of Raman bands were originated from peptides and polysaccharides, which are well known as the bacterial surface components [97
]. This demonstrated that TERS can be used to investigate the more complex biological systems such as bacteria.
Another previous AFM-TERS research of pathogens was tobacco mosaic virus (TMV) which appeared as a rod-like capsid made by one structural protein and one single RNA molecule. In this work, the rod-like structure of a single TMV particle was characterized and four TERS spectra at different positions were obtained through approaching the TERS tip onto the sample surface [98
]. The results indicated that there were some slight differences in the four Raman bands corresponding to the coat protein and RNA of the single TMV. These differences were attributed to the different orientations of the molecules, and might also be caused by moving the TERS probe near to the surface of the sample from one point to another.
In recent years, Bacillus subtilis spores were also investigated by combining TERS with principal components analysis (PCA) [99
]. In this work, the TERS mapping of the spore was analyzed, which provided some important information about the component distribution of the spore ridges (see Figure 6
). These results showed the first and crucial step toward the correlative surface-chemistry characterization of spores via nanoscale spectroscopy. The authors further demonstrated that TERS mapping can be connected to AFM-phase images by the combination of PCA and TERS, which could allow one to visually discriminate the relevant chemical distribution of complex biological systems.
With the characteristics of high sensitivity and superior spatial resolution, the application of AFM-TERS in biology has been increasing rapidly. Table 3
exhibits the structures and detection range of the three biomolecules characterized by AFM-TERS techniques. These results demonstrate that the TERS technique moves further into the direct detection of biomolecules at the nanolevel. However, it is also obvious that the more complex biological molecular structure, the less information can be detected in TERS test. Undoubtedly, this indicates that there is much room for improvement in applying AFM-TERS in biology.
One of the ultimate targets in the study of life science is to seek for the relationship between the structure, function and dynamics of biomolecules in the natural environment. This needs setups which can provide the structural information and corresponding chemical information of samples with a higher resolution. In this view, we have presented AFM-TERS as a technique, which can provide topography and corresponding optical information at a nanoscale resolution at the same time. The outstanding performance makes it an important approach for the study of biochemical species such as nucleic acids, proteins, bacteria, viruses and so on. With a metallized probe, a Raman image with a spatial resolution in the 10 nm range can be obtained by AFM-TERS, it opens a new way in DNA direct-sequencing. Beyond that, it can be used to provide the structural images and label-free chemical information of proteins and pathogens. Those relevant works proved that AFM-TERS could be used to investigate the more complex biological systems and their results could offer beneficial guidelines for biomedical research. For example, AFM-TERS with a sub-10 nm resolution can detect the diseases related to the mutations of DNA [23
]. AFM-TERS can also be used to characterize amyloid fibrils, which can be helpful to seek for the reasons of various neurodegenerative diseases [25
]. Furthermore, biomarkers are biochemical indexes which can be used to measure and evaluate the specific features in biological processes, pathogenic processes, and management processes [100
]. Detecting diagnostic markers can help to the identification, diagnosis, remedy and prevention of diseases because it can mark the changes in the structures and functions of organs, tissues, and cells. The markers include various molecules from inorganic salts to biomolecules (such as DNA, RNA, amino acid, protein etc.), which can be characterized by AFM-TERS. AFM-TERS may become a new analysis method for biomarkers in the near future. All above examples prove that AFM-TERS has the potential to pre-diagnose some clinical diseases and shed light on the pathogenesis of diseases at the molecular level.
In recent years, significant progress has been made in the study of biological materials by AFM-TERS. However, further optimization of the TERS technique is necessary in order to make it suitable for a wide range. For example, studying biomolecules in liquid is crucial since their physiological activities can be kept and detected only in native environments. However, AFM-TERS in liquid just emerges and is in the urgent need of being improved. On the other hand, AFM-TERS tips with a more pronounced performance enhancement should be made in order to avoid biological samples damaged by the high powerful laser. In addition, this system is expected to be developed in the characterization of more soft biological samples.