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 biomarkers 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 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 first coming to market. The researchers demonstrated their system in a study published in the Proceedings of the National Academy of Sciences.
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 biomarkers 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.
"By a thousand times more sensitive,'" the author says, "we mean that if we had a vial of blood with only a few of the relevant proteins, we can accurately count those proteins, whereas a traditional test couldn't reliably tell the difference between that 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 we can quantify the number of proteins at concentrations a thousand times less than they can."
The approach works by measuring one protein at a time, by breaking apart the sample into microdroplets, each of which contain either a single protein or none at all. The lab's expertise in microfluidics 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 here we're turning it into tens of millions of yes-or-no questions," the author says. "Digitizing that question brings down the cost of the camera and the surrounding fluid handling equipment, but shifts the problem into how to process tens of millions of those questions, in a way that is reproducible, accurate, inexpensive and portable."
This technology is based on three key innovations: (i) the integration and parallel operation of a hundred droplet generators onto a single chip that operates >100×fasterthanasingledropletgenerator,(ii)theﬂuorescence detection of droplets at >100× faster than conventional in-ﬂow detectionusingtimedomain-encodedmobilephoneimaging,and (iii) the integration of on-chip delay lines and sample processing to allow serum-to-answer device operation.
To demonstrate the power of this approach, authors performed a duplex digital ELISA. They characterized the performance of this assay by ﬁrst using spiked recombinant proteins in a complex media (FBS) and measured a limit of detection, 0.004 pg/mL (300 aM), a 1,000× improvement over standard ELISA and matching that of the existing laboratory-based gold standard digital ELISA system.
They additionally measured endogenous GM-CSF and IL6 in human serum from n = 14 human subjects using our mobile duplex assay, and showed excellent agreement with the gold standard system (R2 =0.96).
The group has previously published on traumatic brain injury markers, and has an ongoing research project with Presbyterian Hospital with brain injury patients. They also have a spin-off company, Chip Diagnostics, based at the Pennovation Center, which aims to produce test kits for early cancer diagnostics and traumatic brain injury.
Detecting picogram quantities of proteins in blood using a mobile platform
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