More sensitive tests would allow scientists to identify pathogens, diseases and specific cellular proteins even when these “biomarkers” are present at levels far below the detection threshold of today’s standard tests. Initial results show polydopamine boosted the accuracy and resolution of these tests for biomarkers of HIV, Zika virus and proteins on cancerous tumors.

“Common bioassays are the real workhorses of laboratory experiments and medical tests,” said , a 91̽»¨professor of bioengineering. “By boosting the sensitivity of these tests, we can enable more accurate medical diagnoses earlier in a disease or condition, and enable more certainty and less waste in the research process.”

Gao led the team that developed this simple modification for many common medical and laboratory assays. They recently their approach — known as enzyme-accelerated signal enhancement, or EASE — in .

EASE centers on the simple addition of two biochemical components, dopamine and horseradish peroxidase, or HRP, at a key step. HRP is a common protein enzyme used to speed up the rate of reactions in biomedical research. Gao and his team discovered that HRP can connect dopamine molecules together to form the polymer chain polydopamine. Polydopamine, in turn, accumulates on the surfaces of reaction vessels, such as small Petri dishes. Once the polydopamine is present, scientists can continue the traditional steps of their protocols, but now with a substantially increased test sensitivity. Gao hopes that this simple modification will mean that scientists and medical professionals can easily incorporate EASE into their common practices and procedures.

“Scientists have been trying to improve the accuracy of these common tests for decades, but solutions often involve entirely new protocols or costly pieces of equipment,” said Gao. “Understandably, researchers can be reluctant to invest in unfamiliar protocols or expensive new equipment — but EASE is a simple addition to tried-and-true assays. It’s like a software upgrade, instead of changing your operating system.”

These assays include some of the most common medical and laboratory tests, such as , , and . Some of these assays have been used for decades to help hospitals and doctors detect signatures of a disease, ailment or other conditions by looking at a patient’s blood, other body fluids or cells. Depending on the test, these telltale signs could be pieces of a bacteria or virus, a chemical, antibodies made by white blood cells, a hormone or even pieces of DNA.

But if these compounds are present at extremely low levels, diagnostic tests can miss them and return inaccurate medical information. By increasing sensitivity, EASE reduces uncertainty and even increases the amount of information these tests can provide. For example, the team used EASE to detect the presence of Zika virus in the placental tissues of primates. But EASE made the assay so sensitive that they were able to see which types of cells within the placenta were infected with Zika, Gao said.

As often happens in research, Gao and his team did not originally set out to solve this problem. Polydopamine was originally isolated from mussels decades ago, and researchers already knew that the substance can react with proteins. But the only protocol they had to form polydopamine necessitated a passive, time-consuming protocol. Lead author Junwei Li, a 91̽»¨doctoral student in materials science and engineering, was using this approach to coat nanoparticles with polydopamine. But Li noticed that HRP can react with dopamine to form polydopamine, and that this approach is substantially faster than existing methods to make polydopamine.

The scientists don’t fully understand why adding polydopamine boosts the sensitivity of these bioassays, and future research could elucidate the mechanism. But Gao’s focus is on applying EASE to even more diagnostic tests and diseases.

“EASE has potential to solve real, long-standing problems in research and medical tests,” said Gao.

91̽»¨co-authors are pharmacology doctoral student Madison Baird; research scientists Michael Davis and Wanyi Tai; , associate professor of pharmacology and psychiatry and behavioral sciences; , professor of obstetrics and gynecology; , professor of immunology; and , associate professor of pediatrics. An additional co-author is with the Fred Hutchinson Cancer Research Center. Adams Waldorf is also a faculty member with the Sahlgrenska Academy in Sweden, and Rajagopal is also associate professor at the Seattle Children’s Research Institute. The research was funded by the National Institutes of Health, the 91̽»¨ and the Howard Hughes Medical Institute.

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For more information, contact Gao at xgao@uw.edu or 206-543-6562.

Grant numbers: R21CA192985, R01AI100989, AI083019, AI104002, AI060389.

Three scientists at the 91̽»¨ have proposed a way to speed up this waiting game. Their solution, reminiscent of the magic behind washing machines, could reduce wait times to a fraction of what they once were. in the journal , biological assays that once took hours could instead take minutes.

“These are very common assays,” said , a 91̽»¨associate professor of bioengineering and senior author on the paper. “Most scientists were willing to wait hours and hours because they had no choice.”

Many of today’s biological assays use molecules such as antibodies to detect specific types of cellular proteins or pieces of DNA. These “detector molecules” only bind to specific targets, such as a certain class of cellular proteins, and include additional components such as nanoparticles or dye molecules to emit light if they successfully bind. These assays have revealed where different proteins are found in cells and helped diagnose diseases.

But these tests take hours or days to complete. The detector molecules, suspended in a fluid, float around while their targets — whether cellular proteins or pieces of DNA — are adhered to the hard, flat surface of a small plate or petri dish. While bulky detector molecules close to the surface can easily find and bind to their targets, molecules further up in the fluid column move slowly due to their size. It can take hours for enough detector molecules to diffuse down and bind to their targets to produce a visible color change.

“We call this ‘diffusion limitation,’ and it’s a serious problem since both the antibody and nanoparticles are so large,” said Gao. “People have proposed solutions — like stirring or gently rocking a reaction plate to mix the solution. But when we tested this we saw that stirring and rocking only improved the reaction time by 3 to 5 percent. That’s not enough.”

Gao and his team were prompted to tackle the problem of diffusion limitation after they developed a new staining assay but its long reaction times made their protocol impractical. Inspired by studies of fluid dynamics, they decided to work around the problem of diffusion limitation. Instead of waiting for detector molecules to drift down to the surface of the plate, they simply allowed detector molecules close to the surface to bind. Then, they drained the solution from the plate, mixed it, put it back on the plate and repeated this cycle dozens of times — which they call cyclic solution draining and replenishing.

“In a washing machine, you squeeze water out and put it back in,” said Gao. “Dry and re-soak. Dry and re-soak. This is exactly the same mechanism: Drain the fluid completely and then put it back on the plate. That’s much more efficient than simply stirring it around.”

To drain fluid from the plate, they covered the plate with a seal and inverted it. To “re-soak,” they flipped the plate upright again. The flipping action helped mix the detector molecules in the fluid, which sped up the total reaction time.

They tested cyclic solution draining and replenishing with two types of antibody staining techniques, and . Reaction times for both were cut substantially with this drain and re-soak approach. In one case, what was once a one-hour incubation time was cut to just seven minutes. Though sealing and flipping the plate, which they accomplished mechanically, might be impractical for other tests, there are other ways to “drain” a plate.

“We used gravity because we wanted to show that draining would work,” said Gao. “But you could use air bubbles or centrifugation to drain as well, for example. There are lots of possibilities.”

If so, this simple work-around for the problem of diffusion limitation could slash waiting times for experiments. This would also impact other fields, reducing the wait times for medical test results to come back or speed up chemical engineering protocols.

“Really, this was a common problem that no one before had made a link to. But here we have, and it’s so simple,” said Gao. “When we prepare tea, we don’t let it sit there or shake the cup. We repeatedly lift, drain the tea bag, then lower it into the hot water. That’s what we’ve done here.”

Gao was joined on the paper by lead co-authors Junwei Li and Pavel Zrazhevskiy. The work was funded by the National Institutes of Health, the National Cancer Institute, the Howard Hughes Medical Institute and the 91̽»¨.

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For more information, contact Gao at xgao@uw.edu or 206-543-6562.

Grant numbers: R01CA131797 (NIH), R21CA192985 (NIH) and T32CA138312 (NCI).

A 91̽»¨ team has developed a new method for color-coding cells that allows them to illuminate 100 biomarkers, a ten-time increase from the current research standard, to help analyze individual cells from cultures or tissue biopsies. The is published this week (March 19) in .

“Discovering this process is an unprecedented breakthrough for the field,” said corresponding author , a 91̽»¨associate professor of bioengineering. “This technology opens up exciting opportunities for single-cell analysis and clinical diagnosis.”

The research builds on current methods that use a smaller array of colors to point out a cell’s biomarkers – characteristics that indicate a special, and potentially abnormal or diseased, cell. Ideally, scientists would be able to test for a large number of biomarkers, then rely on the patterns that emerge from those tests to understand a cell’s properties.

The 91̽»¨research team has created a cycle process that allows scientists to test for up to 100 biomarkers in a single cell. Before, researchers could only test for 10 at a time.

The analysis uses quantum dots, which are fluorescent balls of semiconductor material. Quantum dots are the smaller version of the material found in many electronics, including smartphones and radios. These quantum dots are between 2 and 6 nanometers in diameter, and they vary on the color they emit depending on their size.

Cyclical testing hasn’t been done before, though many quantum dot papers have tried to expand the number of biomarkers tested for in a single cell. This method essentially reuses the same tissue sample, testing for biomarkers in groups of 10 in each round.

“Proteins are the building blocks for cell function and cell behavior, but their makeup in a cell is highly complex,” Gao said. “You need to look at a number of indicators (biomarkers) to know what’s going on.”

The new process works like this: Gao and his team purchase antibodies that are known to bind with the specific biomarkers they want to test for in a cell. They pair quantum dots with the antibodies in a fluid solution, injecting it onto a tissue sample. Then, they use a microscope to look for the presence of fluorescent colors in the cell. If they see particular quantum dot colors in the tissue sample, they know the corresponding biomarker is present in the cell.

After completing one cycle, Gao and co-author Pavel Zrazhevskiy, a 91̽»¨postdoctoral associate in bioengineering, inject a low-pH fluid into the cell tissue that neutralizes the color fluorescence, essentially wiping the sample clean for the next round. Remarkably, the tissue sample doesn’t degrade at all even after 10 such cycles, Gao said.

For cancer research and treatment, in particular, it’s important to be able to look at a single cell at high resolution to examine its details. For example, if 99 percent of cancer cells in a person’s body respond to a treatment drug, but 1 percent doesn’t, it’s important to analyze and understand the molecular makeup of that 1 percent that responds differently.

“When you treat with promising drugs, there are still a few cells that usually don’t respond to treatment,” said Gao. “They look the same, but you don’t have a tool to look at their protein building blocks. This will really help us develop new drugs and treatment approaches.”

The process is relatively low-cost and simple, and Gao hopes the procedure can be automated. He envisions a chamber to hold the tissue sample, and wire-thin pumps to inject and vacuum out fluid between cycles. A microscope underneath the chamber would take photos during each stage. All of the images would be quantified on a computer, where scientists and physicians could look at the intensity and prevalence of colors.

Gao hopes to collaborate with companies and other researchers to move toward an automated process and clinical use.

“The technology is ready,” Gao said. “Now that it’s developed, we’re ready for clinical impacts, particularly in the fields of systems biology, oncology and pathology.”

The research was funded by the National Institutes of Health, the U.S. National Science Foundation, the U.S. Department of Defense, the Wallace H. Coulter Foundation and the UW’s Department of Bioengineering.

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For more information, contact Gao at 206-543-6562 or xgao@uw.edu. He will be unavailable for interviews by phone or email on Wednesday, March 20.