Although undetectable by the naked eye, the molecular processes being carried out within cells and tissues of the human body have significant consequences for disease risk and treatment. Scientists have developed ways to infer the status of these activities by measuring specific proteins – often called biomarkers – in blood and other bodily fluids. Unfortunately, most laboratory techniques require vials of blood to obtain information about just a few of these tiny proteins – an unappealing prospect for patients and prospective research participants.
This conundrum caused considerable frustration for Dr. Terry Phillips, now a researcher at the National Institute of Biomedical Imaging and Bioengineering (NIBIB). Phillips wanted to examine samples from newborn babies for signs of inflammation. Elevated levels of inflammatory proteins suggest the presence of disease and infection and may be indicative of future health problems. However, Phillips could obtain information about only a few proteins at a time and realized he needed a new tool. “We had to develop microsystems that could measure multiple biomarkers from which we could infer what was happening in the immune system,” he says.
As an immunochemist, Phillips knew how useful antibodies could be for extracting molecules of interest from biological samples. He decided to develop an apparatus to carry out recycling immunoaffinity chromatography (RIC), which refers to the separation of multiple molecules of interest from a single sample using highly selective antibodies. Phillips painstakingly assembled a series of glass tubes, each filled with an antibody able to recognize a protein involved in inflammation. By manually turning valves that linked the tubes, he transferred samples from one glass tube to the next. As the sample flowed by, each antibody extracted its target protein. Phillips then labeled the target proteins with a dye and measured them. The approach worked – 30 proteins could be measured in the equivalent of 1 drop of blood, giving Phillips a much fuller picture of inflammatory activity. However, the process was tedious, making it impractical for large-scale research.
Several years after his initial breakthrough, Phillips has nearly perfected the technology. He now modifies commercially available microfluidics chips to carry out RIC. These chips are fully automated and even more efficient, requiring the equivalent of less than 1 percent of a drop of blood to measure 30 proteins. Furthermore, once built, the chips can be used to evaluate as many as 200 samples before they need to be cleaned and the antibodies reloaded.
Researchers Get More Answers from Less Sample
The RIC microchip allowed Phillips to learn more about inflammation in newborns, but the technology has implications far beyond any individual research project. “We can put any antibody you’d like in the chip,” Phillips says. “As long as you can get antibodies and see how good they are, we can build a chip for anything.” This tantalizing potential has attracted the attention of several scientists. Together with biomedical technician Ed Wellner, Phillips has designed and built microarrays for a number of cutting-edge research studies.
Dr. Giovanni Cizza of the National Institute of Diabetes and Digestive and Kidney Diseases and Dr. Esther Sternberg of the National Institute of Mental Health are part of a group interested in inflammation in women with a history of depression. A few previous studies have attempted to characterize inflammatory proteins called cytokines in this population, but because of limitations in blood volume only a few of these biomarkers could be measured. “That’s not good,” explains Cizza, “Because cytokines act in cascades, and so measuring only a few is really providing only a partial picture.”
Phillips and Wellner crafted a microchip to measure the cytokines of interest in minute quantities of blood collected continuously over 24 hours. When Sternberg asked whether the method could also be used to analyze sweat, Phillips agreed. The research team was able to collect a number of important biomarkers using “sweat patches.” These patches, which are much like an adhesive bandage, stick to the skin and absorb perspiration excreted from the body. Unlike a blood draw, this unobtrusive and noninvasive method allows samples to be collected while patients carry out normal daily activities. Using the customized array, Cizza, Sternberg, and colleagues discovered that women with major depressive disorder have increased levels of several proinflammatory and stress-related proteins. The ability to detect and monitor these proteins may help doctors identify patients at high risk for developing inflammation-related diseases such as cardiovascular disease, diabetes, and osteoporosis, all of which are prevalent in people with depression. Dr. Sternberg reflects on the contributions of Dr. Phillips to this study: “We needed his extremely sensitive and specific methods in order to do this. It just could not have been done without his techniques.”
The ability of the microchip to analyze multiple proteins in very small samples is opening doors for other researchers as well. The technology is extremely helpful for laboratory scientists working with small animal models or low-density cell culture. It also makes it feasible to extract meaningful data from valuable archival material, such as blood samples collected from newborn babies.
Looking Forward to Clinical Applications
So far, the RIC microchip has been used only for research applications, but it is easy to envision how the technology could transform patient care. “I think it is going to be quite a useful tool for the future,” Phillips states excitedly. “I certainly don’t see any limitations to it at the moment.” For example, the microchips may be an effective way to collect molecular “fingerprints” relevant to a number of physiological processes and clinical features, which could be used to diagnose disease or predict response to therapy. The Phillips lab is also working to make the chip and its reader portable so that it could be used for point-of-care analysis as well as in hospital settings.
This research is supported by the intramural program of the National Institute of Biomedical Imaging and Bioengineering.
Phillips, TM. Multi-analyte analysis of biological fluids with a recycling immunoaffinity column array. J Biochem Biophys Methods. 2001; 49(1–3):253–62.
Cizza G, Marques AH, Eskandari F, et al. Elevated neuroimmune biomarkers in sweat patches and plasma of premenopausal women with major depressive disorder in remission: the POWER study. Biol Psychiatry. 2008;64(10):907–11.