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Raman optical tweezers (ROT) as a label-free technique plays an important role in single-cell study such as heterogeneity of tumor and microbial cells. Herein we designed a chip utilizing ROT to isolate a specific single cell. The chip was made from a polydimethylsiloxane (PDMS) slab and formed into a gourd-shaped reservoir with a connected channel on a cover glass. On the chip an individual cell could be isolated from a cell crowd and then extracted with ∼0.5 μL of phosphate-buffered saline (PBS) via pipet immediately after Raman spectral measurements of the same cell. As verification, we separated four different type of cells including BGC823 gastric cancer cells, erythrocytes, lymphocytes, and E. coli cells and quantifiably characterized the heterogeneity of the cancer cells, leukocyte subtype, and erythrocyte status, respectively. The average time of identifying and isolating a specific cell was 3 min. Cell morphology comparison and viability tests showed that the successful rate of single-cell isolation was about 90%. Thus, we believe our platform could further couple other single-cell techniques such as single-cell sequencing and become a multiperspective analytical approach at the level of a single cell.

A novel anti-cancer drug sensitivity testing (DST) approach was developed based on in vitro single-cell Raman spectrum intensity (RSI). Generally, the intensity of Raman spectra (RS) for a single living cell treated with drugs positively relates to the sensitivity of the cells to the drugs. In this study, five cancer cell lines (BGC 823, SGC 7901, MGC 803, AGS, and NCI-N87) were exposed to three cytotoxic compounds or to combinations of these compounds, and then they were evaluated for their responses with RSI. The results of RSI were consistent with conventional DST methods. The parametric correlation coefficient for the RSI and Methylthiazolyl tetrazolium assay (MTT) was 0.8558 ± 0.0850, and the coefficient of determination was calculated as R2 = 0.9529 ± 0.0355 for fitting the dose–response curve. Moreover, RSI data for NCI-N87 cells treated by trastuzumab, everolimus (cytostatic), and these drugs in combination demonstrated that the RSI method was suitable for testing the sensitivity of cytostatic drugs. Furthermore, a heterogeneity coefficient H was introduced for quantitative characterization of the heterogeneity of cancer cells treated by drugs. The largest possible variance between RSs of cancer cells were quantitatively obtained using eigenvalues of principal component analysis (PCA). The ratio of H between resistant cells and sensitive cells was greater than 1.5, which suggested the H-value was effective to describe the heterogeneity of cancer cells. Briefly, the RSI method might be a powerful tool for simple and rapid detection of the sensitivity of tumor cells to anti-cancer drugs and the heterogeneity of their responses to these drugs.

Drug resistance and heterogeneous characteristics of human gastric carcinoma cells (BGC823) under the treatment of paclitaxel (PTX) were investigated using single-cell Raman spectroscopy (RS). RS of normal and drug-resistant BGC823 cells (DR-BGC823) were collected and analyzed using arithmetic, statistic and individual spectrum analysis. The dynamic effects of paclitaxel (PTX) in normal and DR-BGC823 cells were evaluated dynamically. The RS intensity changed with PTX over time and produced distinct different results for the two types of cells. The average RS intensities of the normal BGC823 cells initially decreased and then increased under PTX treatment after 24 hours. In contrast, upon exposure to PTX, the average intensity of the DR-BGC823 cells initially increased within 12 hours and then gradually decreased and approached a steady state. The temporal variation of the typical component in the cells was analyzed by comparing the ratios between Raman bands. More importantly, the heterogeneous characteristics of the BGC823 cells under PTX treatment were quantified and clustered using hierarchical trees combined with RS intensity changes. The 'outlier' cells related to drug resistance were discriminated. The heterogeneity of the normal BGC823 cells under drug treatment gradually appeared over time, and was evaluated with the eigenvalues of principal component analysis (PCA). Our study indicates that single-cell RS may be useful in systematically and dynamically characterizing the drug response of cancer cells at the single-cell level.