One of the frontiers of medical diagnostics is the race for more sensitive blood tests. The ability to detect extremely rare proteins could make a life-saving difference for many conditions, such as the early detection of certain cancers or the diagnosis of traumatic brain injury, where the relevant bio-markers only appear in vanishingly small quantities. Commercial approaches to ultrasensitive protein detection are starting to become available, but they are based on expensive optics and fluid handlers, which make them relatively bulky and expensive and constrain their use to laboratory settings.
Knowing that having this sort of diagnostic system available as a point-of-care device would be critical for many conditions, especially traumatic brain injury, engineers at the University of Pennsylvania have developed a test that uses off-the-shelf components and can detect single proteins with results in a matter of minutes, compared to the traditional workflow, which can take days. Using a standard cellphone camera and a set of strobing LED lights, combined with their lab's microfluidic droplet generators, the team has developed a system that is a thousand times more sensitive than the standard protein assay, is handheld, and considerably less expensive than the current state-of-the-art single-protein tests just coming to market.
The standard protein detection assay, ELISA, involves attaching antibodies to the proteins in question, then measuring how much the sample's color changes in response to enzymes linked to the antibodies. This process is fast and simple enough to be incorporated in point-of-care devices, like home HIV tests, but only works when the proteins are in large concentrations.
There are currently very few bio-markers for traumatic brain injury because very few of the protein markers of those injuries make their way through the blood-brain barrier. Medical researchers have only recently confirmed that any such markers could be used for a blood test, and given their ultra-low concentrations, that test would need to be much more sensitive than the standard ELISA array. A traditional test couldn't reliably tell the difference between a vial of blood and one with none of the protein in it. As you keep increasing the number of proteins, the traditional test will eventually be able to detect them, but this new system can quantify the number of proteins at concentrations a thousand times less. The approach works by measuring one protein at a time, by breaking apart the sample into micro-droplets, each of which contain either a single protein or none at all.
The Penn lab has produced microchips etched with hundreds of microdroplet generators, all working in parallel. Normally, you'd have to measure very precisely how much a sample changes color or fluoresces, but instead, they're turning it into tens of millions of yes-or-no questions. Digitizing that question brings down the cost of the camera and the surrounding fluid handling equipment and shifts the problem into how to process tens of millions of those questions, in a way that is reproducible, accurate, inexpensive and portable.
While an off-the-shelf camera can detect whether a microdroplet contains a fluorescent-marker-bound protein or not, the big challenge was to speed up the process. Existing digital droplet detectors line the droplets up so they can be measured one at a time. Such systems are accurate, but bulky and expensive. They also have limited throughput, because of the need to look at millions of droplets one at a time. A thousand droplets a second, the throughput of conventional technologies, is still slow if you need to measure 50 million.
Rather than having a single channel, the researchers flow droplets into hundreds of channels that pass by the camera at the same time. The bottleneck, however, is how fast a camera can capture the data. Conventionally, that wouldn't work since the exposure time you'd get from a regular camera is such that the signals from two droplets next to each other would overlap. A cellphone camera takes about a hundred images a second, which is far too slow to be useful to resolve these droplets. But you can use that camera if the light source you're using to illuminate the droplet strobes a thousand times faster than the framerate of the camera. The trick was to encode this strobing light with a signal that would enable teasing apart one microdroplet from its neighbors. The light is strobed in a very specific pattern that never repeats itself, which is a technique borrowed from radar. As the signals go across the screen they get imprinted with a barcode. So even though they overlap with one another, they can still be identified by which strobe pulse illuminated each droplet.