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Bar code measures protein concentrations quickly and accurately

BioPhotonics
Feb 2009
Amanda D. Francoeur, amanda.francoeur@laurin.com

PASADENA, Calif. – In a fast-paced world, waiting several days for the results of a blood test can be extremely frustrating, especially if it’s for something important. Now researchers at California Institute of Technology have devised a bar-code chip that will end the wait and reduce blood sample sizes, all while testing for proteins that may indicate illnesses such as cancer or heart disease. The results were published in the December 2008 issue of Nature Biotechnology.

SNbarcode_Fig-1.jpg
A main channel directs blood through the integrated blood bar-code chip. Smaller channels positioned at 90° angles from the main channel skim pure plasma from whole blood. The plasma is transferred into the bar-code section of the device, where it is assayed for 12 specific protein concentrations.


“This is a generic platform to analyze health and disease conditions of multiple organs simply through blood,” said research team member Dr. Rong Fan of the NanoSystems Biology Cancer Center at Caltech.

It’s called an integrated blood bar-code chip, and it can measure concentrations of a dozen proteins using a single pinprick’s worth of blood from a patient’s finger, producing results in less than 10 minutes.

In contrast, a standard blood test consists of filling vials with samples taken from a patient’s arm and sending the specimens to a lab. Results usually aren’t available until days after the testing because the samples must be centrifuged to separate the whole blood cells from the plasma, the liquid portion of the blood, which is assayed for specific proteins. The process is also expensive; a diagnostic kit detecting a single protein costs around $50.

Setup and function

The chip is a glass substrate covered with silicone rubber; the surface of the device is molded to contain a microfluidics circuit – microscopic channels that separate out the plasma for testing. The device, which is no bigger than a microscope slide, can test a blood sample from eight to 12 patients simultaneously and can detect multiple proteins at a time from each specimen.

Once the blood sample enters the chip, 15 percent of the plasma is extracted from the whole blood through narrow high-resistance channels branching off from a bigger, low-resistance main channel, and the plasma is transferred into the bar-code portion of the device. The whole blood is directed into a waste outlet. Only a small sample of blood – a few microliters – is necessary to yield the few hundred nanoliters of plasma required.

The plasma is then immunoassayed for protein biomarkers by a DNA-encoded antibody library (DEAL) bar code consisting of a series of lines. Each line is 20 μm wide and coated with a different antibody. The antibodies tested include prostate-specific antigen (PSA), granulocyte-macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF-α), all of which are sensitive to anomalies that may be present in a sample.

As the plasma flows over the bar code, specific proteins are caught on the lines by the antibody. When the plasma has been developed through the bar code, the lines emit a red fluorescent light that varies in intensity, depending upon the amount of protein present.

“In this work, we had 12 bars assigned for detecting 12 different target proteins using a red fluorescent molecule (Cy5),” Fan said. “The combination of multiple stripes gives rise to a protein profile – for example, in the blood of a patient.”

The reason for including multiple antibody-layered lines is that many human diseases are heterogeneous and cannot be revealed by a single biomarker. According to Fan, cancer is particularly hard to determine because it has multiple origins. “Cancer is never a single disease but actually encompasses a large number of disease states due in part to different genetic mutation patterns,” he said. “Therefore, multivariate analysis is needed to identify the subtypes and allow patients to be correctly stratified to enable personalized treatment,” he added.

Precision and distinction

Protein concentrations are finely tuned biomarkers potentially indicating disorders and their severity. To measure protein concentration specifically via the integrated blood bar-code chip, the researchers measured human chorionic gonadotropin (hCG), a hormone induced by pregnancy, and concluded that the chip could gauge substantial increases in hCG. They also compared blood samples from 22 patients who had breast or prostate cancer, establishing that the combinations and concentrations of biomarkers differ between the two types of cancer.

SNbarcode_Fig-2.jpg
The bar-code lines, coated with antibodies, appear fluorescent red when specific proteins from a sample of blood plasma are caught on the bars. Brighter fluorescence indicates a higher concentration of protein.


Furthering the study, the scientists determined that protein concentrations vary not only by disease but also by individual. For instance, two women at different stages of the same type of cancer will have distinguishable sets of proteins. The bar code also registers protein changes in a patient over the course of therapy.

An advantage of the chip’s acute sensitivity is that it will enable the development of individual treatment regimens. It also could be helpful in collecting data from a patient undergoing treatment and monitoring a drug’s effectiveness over time.

According to lead researcher Dr. James Heath, the chip is being tested in clinical practice for patients with glioblastoma, a malignant brain tumor that affects the central nervous system. Healthy patients are being studied for protein composition variations from exercising and dieting.

The researchers’ goal is to create an integrated blood bar-code chip that measures 100 different proteins from a single blood sample, which they predict will take about a year to develop.


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