We present an in situ mRNA extraction platform to quantify marker-genes' expression levels of single target cells within high-density microfluidic trapping arrays. This platform enables single-cell transcriptomic analysis to reveal in-depth information of cellular mechanisms and population heterogeneity. Although microfluidic technology enables the automation of single-cell sorting, trapping and identification, most developed microfluidic devices are closed off and prevent single-cell access by external analytical equipment. Besides, cell lysing is usually required for mRNA extraction. In our platform, cells are trapped individually in a microwell array sealed by a 1 μm-thick polydimethylsiloxane (PDMS) membrane, and a modified atomic force microscopy (AFM) probe—a dielectrophoretic nanotweezer (DENT)—penetrates through the membrane and extracts mRNA molecules from a single cell by dielectrophoresis. The single-cellular expression levels of 3 housekeeping genes from HeLa cells were analyzed quantitatively based on the quantification of the extracted mRNAs, and the probed cells remained viable when the applied alternating-current (AC) voltage was lower than 1.5 Vpp during mRNA probing. We also performed in situ mRNA isolation from a mixture of SK-BR-3 and U937 cells, mimicking a blood sample that underwent primary enrichment of circulating tumor cells (CTCs), and evaluated various marker-genes' expressions. This integrated platform combines the non-destructive and precise-control of a single-cell mRNA probe with sealed microfluidic systems' capability of upstream sample processing and downstream multifunctional analysis to enable a versatile and powerful tool for biomedical research.

A billion-fold increase in the Raman signal over conventional tip-enhanced Raman spectroscopy/microscopy (TERS) is reported. It is achieved by introducing a stimulating beam confocal with the pump beam into a conventional TERS setup. A stimulated TERS spectrum, closely corresponding to its spontaneous TERS counterpart, is obtained by plotting the signal intensity of the strongest Raman peak of an azobenzene thiol self-assembled monolayer versus the stimulating laser frequency. The stimulated TERS image of azobenzene thiol molecules grafted onto Au ⟨111⟩ clearly shows the surface distribution of the molecules, whereas, when compared to the simultaneously recorded surface topography, it presents an image contrast of different nature. The experimentally obtained stimulated gain is estimated at 1.0 × 109, which is in reasonable agreement with the theoretically predicted value. In addition to the signal increase, the signal-to-noise ratio was 3 orders of magnitude higher than in conventional spontaneous TERS. The proposed stimulated TERS technique offers the possibility for a substantially faster imaging of the surface with respect to normal TERS.Keywords: self-assembled monolayer; stimulated Raman; surface chemical imaging; tip-enhanced Raman spectroscopy

Selective messenger RNA (mRNA) profiling of transfected breast cancer gene expression in a living cell is demonstrated. Atomic force microscope (AFM) probe tips are structurally modified to create a dielectrophoretic force that attracts mRNA molecules within the cell nucleus. The tip end is chemically modified to hybridize only to the target mRNA from a pool of molecules within the nucleus. We successfully combined this scheme with standard assay techniques to develop an assay technology that can be used for early disease detection and basic studies in cell biology.