Technology

Cellular Highways instruments will be powered by a new type of microfluidic cell sorter, VACS: vortex actuated cell sorting (patent pending).

Like in FACS, a stream of fluorescently labelled cells is measured optically, and sorting decisions are made in real time. In our technology, cells are deflected individually by a new type of sorting mechanism: a microscopic inertial vortex in the flowing medium that is created by a thermal vapour bubble. Our system requires no side channels, no sheath fluid and no aerosols, only one input and two outputs (sorted cells and waste) on a microfluidic chip.

By multiplexing this technology, we will be able to deliver far higher sort rates than existing platforms, and thus enable new diagnostic and therapeutic applications.

Cell sorting with an inertial vortex

VACS uses a thermal vapour bubble actuator, also known as a bubblejet actuator, to deflect cells. Thermal vapour bubble actuators have some attractive properties for microfluidic applications. The actuator is a very small (~100 µm) electrical microresistor – much smaller than ultrasonic actuators, for example – and can be made by standard MEMS manufacturing processes on a glass or silicon substrate. It is driven directly by an electrical pulse without the need for external transducers, such as external electromagnets or piezo-actuators.

The displacement produced by a single bubblejet-style actuator is extremely small and short-lived, however. Using bubblejet-style actuators to deflect cells is therefore a significant challenge. To deflect a cell, it is necessary to create a displacement greater than the diameter of the cell. Moreover, the time scale of the displacement needs to match the transit time of the cell through the junction between the input and output channels. If the displacement is too short-lived, cells are pushed one way and then the other before they enter the sorting channel, and are therefore not sorted.

Previous researchers have solved this problem by: (1) placing the actuator in a side channel to focus the displacement in a smaller area, (2) using many bubblejet-style actuators in parallel to increase the displacement amplitude and duration, or (3) employing a high-power laser rather than an electrical microheater to create the bubble. However, these approaches add complexity to the sorter chip and make it difficult to envisage a practical, low-cost or multiplexable cell sorting instrument.

We decided to position a single bubblejet-style actuator at the side of the main sorting channel, thus avoiding the need for additional side channels. At the same time, the actuator is far enough from passing cells so that cells to be sorted don’t get ‘singed’ or perturbed by the thermal event associated with the vapour bubble.

With the bubblejet actuator positioned at the side of the main channel, we then required a way of converting the small, short-lived displacement generated by the vapour bubble into a large, lasting cell displacement.

We achieved this by creating a pattern of gentle bends as well as a sharp edge in the channel. In this configuration, the thermal vapour bubble creates an inertial vortex at a well-defined position in the stream that flows downstream with the cell of interest, thus causing a permanent cell displacement.

Thanks to accurate simulations of the fluid dynamics of this system, we were able to design and test the inertial vortex in silico. The animations below show how an inertial vortex deflects individual cells.

Deflection of a cell in a stream by an inertial vortex, showing the total flow (steady stream left to right, from input to outputs).

The same simulation, showing the transient flow only: the creation of the inertial vortex and its deflection of a single cell can be seen clearly.

We then built a complete fluorescence-activated cell sorting system, implemented in a demonstrator rig, to show that inertial vortex sorting works. The sorting process is too fast for even high-speed imaging, but a strobe light allowed us to visualise and verify sorting events.

Experimental verification of the sorting system by double-strobe imaging. The video shows a compilation of independent double-flash strobe frames (rather than a real-time video). In each frame, the cell of interest is detected upstream by laser fluorescence. If the fluorescent signal is within pre-defined thresholds, a double image is made of the deflection event, by flashing the strobe light twice: once in the location of the thermal vapour actuator, when the cell is before the sort junction, and once when the cell has travelled beyond the sort junction, to check which output channel it has entered. We then count correct events using image analysis.

Advantages of VACS

Our core technology is fast, small, suitable for multiplexing and highly manufacturable.

In the demonstrator rig, VACS achieves the following:

  • Eqivalent droplet rate / switching speed of 43,000 / second
  • Maximum cell deflection rate of 5000 / second (we believe we can raise this to around 12,000 / second with optimisation)
  • Error rates for 10 µm particles: < 0.01% (false positives), < 0.5% (false negatives)
sortwindowhistogram
The sort mask (or sort window) is the key measure of performance of a particle-sorting technology. This function of time or space along the stream shows the basic ability of the technology to pick particles precisely and avoid false positives or false negatives. Our mask width is 52 μm (or 23 μs).

Our core technology is also extremely small. The dimensions of the device on chip is 1 mm x 0.25 mm, without the need for an external actuator or side channels. Therefore, our core technology is attractive for multiplexing on chip or integration with lab-on-chip workflows.

Finally, our basic cell sorting chips are highly manufacturable because they are made with standard MEMS processes and materials and incorporate only a single layer of microfluidics.

device
Our core device is both small and simple, incorporating only an input, two outputs, no side channels, and a 250-µm-wide device in the centre. The actuator and electrical connections are made by standard MEMS thin film processing techniques on a glass substrate, and a single microfluidics layer is bonded to the glass.

Our demonstrator rig

Our laboratory demonstrator rig incorporates a single laser (488 nm), lenses, filters, detectors and fast electronics to measure fluorescence, forward scattering and backscattering to decide which cells to sort in real time.

opticsjig
Our custom-built optical analyser and control system, for testing the inertial vortex sorter chips. The system incorporates laser stimulation, fluorescence, forward scatter and back scatter, strobe imaging, FPGA real time control, PC monitoring. The sorter chips are fed by a single syringe pump.

Next step: multiplex sorting

We have started work on the next generation of our technology: a 16X multiplex cell sorter. The 16X system is expected to sort living cells much faster than any device has ever achieved before. The instrument is based on the same principles, but consists of 16 Inertial Vortex Sorters on a 4 x 4 grid of pitch 1 mm.

We are aiming for:

  • Cell processing rate: 2.5 billion / hour
  • Peak cell sort rate: 0.7 billion / hour
The next generation of the technology: we are working on a 16X multiplex sorter, i.e. 16 inertial vortex sorters on a 4 x 4 grid of 1 mm pitch.