Non-invasive and non-destructive identification of different cell types allow for the early stage diagnosis and lead to more efficacious potential treatment of various human diseases. For example, early stage cancer detection enables many more treatment options and potential cure as compared to detection in the later stage of cancer. In this respect, circulating tumor cells (CTCs) in the blood stream have been shown to be a strong indicator of early stage of various cancers. However, separation, capturing and identification of CTCs still possess significant challenges with regarding to their extremely low concentration as well as the inability of traditional methods to characterize them accurately.
The problem of capturing and identifying CTCs is undertaken using two different approaches in our lab: (a) isolation of CTCs from blood using dielectrophoresis and (b) spectroscopic identification of cells using mid-IR plasmon resonant metasurface sensors.
Spectroscopic identification of cells using mid-IR plasmon resonant metasurfaces
Mid-IR spectroscopy is one of the prominent ways of identifying different materials via their fingerprint molecular vibrations. In the past, this has been used for spectroscopically distinguishing cancerous versus non-cancerous tissue. Typically, multiple layers of cells or a complete monolayer of cells is required for performing such a characterization. This limitation on the number of cells creates a large hindrance for adapting this technique for the detection of CTCs, due to their inherently low concentration. It has been previously shown by our group that mid-IR spectroscopy performed using plasmon resonant metasurfaces (Figure 1b,c) allows one to enhance the sensitivity of this technique significantly and we used this approach to accurately characterize a single protein layer. The increase in sensitivity arises from the highly enhanced optical electric fields created near the structures. Furthermore, the metasurface only probes a small region close of the cell membrane due to the rapid decay of the enhanced fields away from the metasurface.
Figure 1. Spectroscopy of biomolecules and cells using plasmonic metasurfaces. a) Fluidic flow chamber to deposit cells on the metasurface. The chamber is mounted under an IR microscope and measured through the substrate while keeping the sensor surface in fluid. b) Cells deposited onto the metasurface (black square). The inset shows an SEM image of the zoomed in area of the metasurface unit cells. c) Artists rendering of metasurface unit cells with biomolecules attached to them. d) Measured absorbance of CCD841 and RKO cells. A bare metasurface is used as reference. Several molecular vibrations can be observed, as well as the difference between the two cell types.
To demonstrate that this technique is viable for cell distinction at a few cell level, we used these metasurfaces for distinguishing between cancerous (RKO) and non-cancerous (CCD841) colon cells. A typical image of metasurfaces with cells on them is depicted in Fig. 1b, where the darker region indicates the metasurface. Figure 1d shows a set of representative mid-IR spectra from the two different cells that clearly show a large difference. The difference in the spectral features between the two cell types can thus be used for identifying them. Note that the spectra were acquired in the aqueous state, which is generally not the case in most of the studies in literature. Finally, from a device perspective, the whole experiment is performed within a flow chamber enclosing the metasurfaces (shown in Figure 1a), which paves the way for automated and rapid identification and characterization of cells.
DEP based cell capturing
One way to improve deposition of cells directly onto the metasurface sensor is to use dielectrophoresis (DEP). Since cells act like dielectric particles, a nonuniform AC electric field can be set up around the metasurface using specific electrode configurations which causes cell movement due to DEP force (proportional to electric field gradient, Figure 2b).
Figure 2. a) Schematic of the experimental set-up to capture CTCs with DEP force. A large ITO plate electrode is placed on one end of a flow channel whereas discrete electrodes run through the metasurface. The ITO electrode and strips are connected to an AC function generator to generate non-uniform AC electric field inside the channel. At the correctly chosen frequency CTCs (purple) will move towards the metasurface sensor and get captured while blood cells (blue) will be repelled and flushed away by continuous stream of fluid. b) Computer simulation of the cross section of the experimental configuration shown in (a) with 4 wires at the bottom and plate electrode at the top. Color shows the magnitude of electric field and arrows the gradient of electric field (proportional to DEP force). At the correctly chosen frequency the direction of the force for blood cells is opposite to the force acting on CTCs.
Attachment of the cells to the sensor surface can be improved by covering the sensor with antibodies. Furthermore, by tuning the electric field frequency and conductivity of the solution, it is possible to capture specific cells while repelling other kinds of cells in a multi-species cell solution. An illustration of such experimental configuration is shown on Figure 2a. Separation of different cell types is especially important while working with blood samples that have very low concentration of CTCs. In the case of CTCs, the separation of tumor and blood cells with DEP is very effective, since those cell types have very different dielectric properties and therefore the frequency of the electric field can be chosen such that CTCs move to the sensor while pushing the blood cells away from it.
- Fano-resonant asymmetric metamaterials for ultrasensitive spectroscopy and identification of molecular monolayers. Chihhui Wu, Alexander B. Khanikaev, Ronen Adato, Nihal Arju, Ahmet Ali Yanik, Hatice Altug, Gennady Shvets; Nature Materials 11, 69–75 (2012)