A team of researchers has developed a barcode chip that they say can measure a broad panel of proteins across a wide dynamic range in 10 minutes or less and with as little as a finger prick’s worth of blood, potentially offering a cheaper and faster diagnostic tool than is currently available.
And as the state of blood-based protein biomarkers remains dubious, the technology could help validate relevant biomarkers and move them to clinical utility, one of the researchers told ProteoMonitor.
The microfluidic technology, called the integrated blood barcode chip, was developed by researchers at the California Institute of Technology, NanoSystems Biology Cancer Center, the Kavil Nanoscience Institute, and the Institute for Systems Biology.
In a paper published Nov. 16 in the online edition of Nature Biotechnology describing the IBBC, the researchers also tested the device’s ability to detect human chorionic gonadotropin, a hormone produced during pregnancy, and biomarkers linked to breast and prostate cancer.
In the paper, the authors said that because blood contains the largest representation of the human proteome of any other fluid or tissue in the body, it is the most important clinical diagnostic medium. Changes in plasma-protein profiles can indicate many diseases, but only a “handful” of plasma proteins are used for diagnostic purposes due to the intrinsic complexity of the plasma proteome, the heterogeneity of human diseases, and the rapid degradation of proteins in blood samples.
Because the IBBC can separate and measure a panel of plasma proteins over a broad concentration of rages and within 10 minutes of sample collection, it could overcome many of the roadblocks associated with the development of blood-based protein diagnostics, the researchers said.
“The concept of the chip is to try to demonstrate that you could do traditional clinical blood proteomics for a very low cost, which is going to be more important as people want to measure more and more proteins out of blood,” James Heath, the corresponding author on the paper, told ProteoMonitor.
In traditional blood tests measuring proteins, researchers use centrifugation to separate whole blood from plasma, and then assay the plasma for specific proteins. According to Heath, a professor of chemistry at CalTech, the process is both labor-intensive and time-consuming, typically taking a few hours to complete. A kit to test for a single diagnostic protein costs $50, he said.
“The main purpose [of the study is to show] that you can take glass and plastic and forget about all the steps of centrifugation and all the time involved in a standard ELISA measurement [and] reduce it down to something very short and still measure a large number of proteins,” he said.
He said the IBBC can now measure about 50 proteins from a finger prick’s worth of blood — typically between 5 and 10 μL — in as few as 5 minutes and for the same cost as measuring a single protein with standard ELISAs. Each IBBC can simultaneously test blood from eight patients.
The researchers are currently working to optimize the technology so that it can measure 100 proteins. The bottleneck, Heath said, is antibody quality, which his group is trying to solve by finding alternative protein-capture reagents.
To be sure, developing a faster and cheaper diagnostic based on protein biomarkers found in blood may be putting the cart before the horse considering that it remains unclear whether any blood-based biomarkers discovered to date have any clinical utility. But Health said that the chip can also be used to validate such candidate markers, therefore pushing them closer to the clinical setting.
“We hope our approach … enables us to not just choose the one protein that you want to measure, but [many leading candidate proteins] and to really rapidly do them across many patients [and] many diseases” in order to find the biomarkers that truly are predictive of disease, Heath said. “It hastens the [validation step]; it makes it much more inexpensive to … test those biomarkers across a broad class of different patient, different diseases, and you could do a whole lot of biomarkers at once.”
Testing with Just a Prick
The chip, about the size of a microscope slide, is made out of a glass substrate covered with silicone rubber. The IBBC surface is molded to contain a microfluidics circuit that collects the blood, separates the plasma from the whole blood, and measures a panel of protein biomarkers.
The researchers exploited the Zweifach-Fung hydrodynamic effect “by flowing blood through a low-flow-resistance primary channel with high-resistance, centimeter-long channels that branch off it at right angles,” they wrote in the article.
“The main purpose [of the study is to show] that you can take glass and plastic and forget about all the steps of centrifugation and all the time involved in a standard ELISA measurement [and] reduce it down to something very short and still measure a large number of proteins.”
Plasma is directed toward the high-resistance channels, while remaining whole blood is directed toward a waste outlet. The glass base of the plasma-skimming channels is patterned with a dense barcode-like array of single-stranded DNA oligomers, and a full barcode is repeated multiple times within a single plasma-skimming channel. Each barcode sequence constitutes a complete assay.
To detect proteins, Heath and his colleagues used the DNA-encoded antibody library technique: DNA-directed immobilization of antibodies was used to convert a pre-patterned ssDNA barcode microarray into an antibody microarray, which provided “a powerful means for spatial encoding,” the authors wrote.
Because only a few microliters of blood is normally sampled from a finger prick, and on-chip plasma separation yields a few hundred nanoliters of plasma, the researchers patterned the ssDNA barcodes at a high density using microchannel-guided flow patterning to increase the chip’s ability to measure a large panel of protein biomarkers from the small volume.
They also coated their glass slides with polyamine, which allow “substantially higher DNA loading” than traditional aminated surfaces “and provide for an accompanying increase in assay sensitivity,” the researchers said. Additionally, IBBC uses 20-micrometer-wide bars spaced at a 40-micrometer pitch, an array density that represents an approximately 10-fold increase over a standard spotted array, “thus expanding the numbers of proteins that can be measured within a small volume.”
To evaluate their chip, they tested its detection range with hCG, a hormone widely used for pregnancy testing and whose concentration increases by five orders of magnitude during pregnancy. The IBBC was able to capture the entire concentration range in a single test, the researchers reported.
Next, they assessed the IBBC with serum collected from 11 patients with breast cancer and 11 with prostate cancer. The chip was able to differentiate the two types of cancers, and within each cancer was able to differentiate concentrations of protein biomarkers.
However, though their work on prostate cancer validated the applicability of the technology, “the statistical accuracy of the PSA barcode assay was not high, revealing only a modest linear correlation” between the ELISA and the DNA-encoded antibody library technique used to develop IBBC, the authors said.
They attributed this to the manual chip-manufacturing process and said that they are currently automating barcode fabrication, assay execution, and image quantification “in an effort to bring statistical uncertainties to within 10 to 20 percent, which would be close to the state of the art.”
Overall, Heath said that that part of the team’s study demonstrated the chips could measure proteins at clinically relevant levels, and it statistically validated the chips relative to standard protein measurements.
He said their work also showed that the technology has potential to stratify cancer patients, though the authors point out their study does so only as a proof of principle. For example, their results for breast cancer can be grouped into three subsets: non-inflammatory; interleukin-alpha positive; and tumor necrosis factor-alpha/granulocyte-macrophage colony-stimulating factor positive. And the prostate cancer patient data were classified into two major subsets based upon the inflammatory protein levels.
While the authors used a common laboratory scanner to read the barcode information on the chip, “it should be very easy to design something like” retail barcode scanners to read the IBBC, Rong Fan, the first author of the study, said in a statement. Heath said he and his colleagues are not involved in developing such an instrument, however.
The IBBC is currently being tested in human clinical trials on patients with glioblastoma and melanoma. They are also building a database containing information on levels of various proteins for healthy patients and how they vary as a function of diet, sleep habits, and other lifestyle factors.
Heath declined to elaborate on the ongoing work but said that the “hypothesis that you get better results from very rapid assays … looks like it’s probably correct, and that you don’t get … cross-reactivity. That becomes much less important when the blood is very, very fresh.”