Uncontrolled growth of the human immunodeficiency virus (HIV) causes patients to develop acquired immunodeficiency syndrome (AIDS). However, about 1 out of every 300 HIV-infected patients doesn’t get AIDS because his or her immune system suppresses the virus. The immune systems of these so-called HIV controllers contain specialized T-cells that restrict growth of the virus so that it is nearly undetectable in their blood. Researchers want to learn why HIV controllers’ T-cells are so effective at limiting HIV’s ability to replicate and are hopeful that understanding how their T-cells work will aid development of effective AIDS vaccines.
To investigate this phenomenon, scientists are turning to bioassays or lab-on-a-chip technologies. One new approach developed by a team at the Massachusetts Institute of Technology (MIT) may allow AIDS researchers to detect the specific proteins in HIV controller T-cells that keep HIV at bay. “No tool has allowed us to do this until now,” says AIDS researcher Bruce Walker, director of the Ragon Institute in Boston, MA. Walker is collaborating with the MIT team to develop new T-cell bioassays.
Developed by MIT’s Patrick Doyle, associate professor of chemical engineering, and former student Daniel Pregibon, the new bioassay system involves three activities: fabricating unique microparticles, mixing the microparticles with patient samples, and scanning the resulting mixture with an ultra-rapid detection system. “Flexibility in how we create our particles will allow us to deliver entirely new classes of assays,” says Doyle. “This is a very scalable, straightforward process. It’s not just pixie dust in my lab.”
To create the microparticles, Doyle and Pregibon borrowed a page from the computer industry and use a photomask technique. In essence, the particles form when a beam of ultraviolet (UV) light passes through a barcode template and a microscope lens and strikes two flowing streams of bio-friendly material called hydrogel. One stream contains probes—DNA sequences and proteins—and the other stream contains fluorescent imaging agents. When the UV light hits the hydrogel, it binds the two streams together and imprints the predetermined barcode on the material.
The resulting microparticles resemble tiny dominos and range in size between 10 and 100 µm (from the size of a red blood cell to the width of a human hair). One side of the particle contains barcodes that will identify a target DNA sequence or protein, and the other side holds probe material that links to a corresponding target in the sample.
To run an assay, researchers first select microparticles with barcodes that match the target components they want to study. They then mix the particles with the sample. In the case of the HIV research, patient blood samples will be mixed with microparticles coded to identify proteins produced by T-cells. During the mixing process, DNA sequences or proteins from the sample migrate to the microparticle and link up with matching DNA sequences and proteins.
To view these connections, the mixture is placed in a high-throughput detector. As the microparticles flow through a laser beam, a fluorescent signal will activate if there is a match between a target in the sample and the DNA or protein on the particle. Computer analysis of the fluorescent signals decodes each particle, identifying the types of target molecules that are linked to the particle and, based on the intensity of the signal, the amount of each target on the particle. Only a very small sample is needed because the detection system can identify target molecules at extremely low concentrations.
The MIT approach advances typical bioassay technology because it uses biocompatible materials, can simultaneously identify and quantify multiple biomolecules, and is inexpensive. Unlike plastic and metal materials that are used to make some microparticles, hydrogel retains water and allows biological reactions to occur in three dimensions, not just on the surface. This increased data capacity improves sensitivity of tests. The domino fabrication process can generate over 1 million individual codes to identify target compounds. Commercially available systems generate about 100 codes. Because the detection system uses a single laser, detector costs stay low.
Diagnostics and Beyond
New research shows that microRNAs—molecules that control protein activity—play a key role in disease progression. Developing a system to detect microRNA could aid diagnostics for cancer and other diseases. Doyle and Pregibon recently applied their technique to identify microRNA associated with lung cancer. Using tissue from lung cancer biopsies, the system took just three hours to identify microRNA molecules with an accuracy of 98 percent. This rapid screening time is an eightfold reduction of time required when using conventional microarray methods.
Pregibon, who co-founded Firefly Bioworks in 2009 to commercialize the technology, is also developing a clinical version of the scanning system. He envisions a portable system that doctors can use in their offices to diagnose disease. Over time, Pregibon predicts the system could become a routine way to monitor a patient’s health. “In the future, diagnosis will be the best form of treatment.”
HIV research and cancer diagnostics are just two applications of the microparticle screening technique. In addition, because the scanning system can detect thousands of biomolecules at one time, it could become a vital tool in developing novel therapeutics by determining drug effects on the body. The MIT fabrication process itself offers drug companies a new approach to producing pharmaceuticals. Current batch-based systems can take weeks to complete because of the multiple steps involved. The MIT approach is continuous and can churn out over 10,000 complete particles in an hour. The bio-friendly particles themselves could find applications in consumer products such as sunscreen or shampoo as more and more manufacturers look to nanotechnology to enhance their products.
This work was funded in part by the National Institute for Biomedical Imaging and Bioengineering.
Chapin SC, Appleyard DC, Pregibon DC, Doyle PS. Rapid microRNA profiling on encoded gel particles with a high-throughput microfluidic scanner. (In press).