(101d) Two-Phase Microfluidic Flow System for the Isolation of Circulating Melanoma Cells Detected Using Photo-Acoustics

The Photoacoustic Effect occurs when cells that contain pigments (such as melanoma cells) absorb incident laser radiation, suffer a thermo-elastic expansion, and as they expand, give rise to a pressure (acoustic) pulse. By detecting these acoustic pulses using a piezoelectric pressure transducer, we have previously been able to detect very low numbers (< 10/ml) of metastatic melanoma cells, which contain the pigmented granule melanin, in samples also containing much large numbers (> 1,000,000/ml) of leucocytes and platelets, which, because they contain no pigment, are “photo-acoustically transparent”. Thus, the Photoacoustic Effect has emerged as a rapid and sensitive method of diagnosing and monitoring Melanoma, the deadliest form of Skin Cancer, at an early stage (before the circulating melanoma cells establish secondary tumors).

Further information about the circulating melanoma cells, such as their molecular biology and gene expression profile can be obtained if these cells can be isolated. But this is a difficult task given their low numbers (< 10/ml) and the high numbers of other cells present (> 1,000,000/ml). We report here, a novel two-phase microfluidic method for isolating our target melanoma cells that is rapid and effective.

When two immiscible fluids (aqueous suspension containing cells, and air, in the present case) are introduced from the two sides of the “horizontal” arm of a T-channel network at constant flow rates and forced to flow together through the same narrow channel (the “vertical” arm of the T-channel), the two fluids, due to the effects of surface tension, spontaneously form alternating “slugs” (droplets that span across the entire cross-section of the channel). Each slug has a volume of  ~10 ul, and contains roughly 10⁴ WBCs and platelets, but only a very small number of these slugs (approximately equal to the number of target cells) contain target cell(s). The slugs of air present on either side of a slug of aqueous solution confine the acoustic signal, and allows each aqueous slug to be individually interrogated for the presence of photoacoustic- positive (melanoma) cells. At a certain distance downstream, the slugs are sorted, with only the photoacoustically-positive slugs collected, and the rest discarded. This step reduces the number of other cells by a factor of 100. The collected solution can be diluted back to the original volume, and the process repeated multiple times, with a similar 100-fold decrease in the number of other cells, till a suspension containing target cells alone, is obtained.  The label-free nature of the isolation technique also eliminates the need for other pre, and post-processing steps that may affect or alter the molecular biology of the targets.