There is a strong interest in the simultaneous detection of a number of different proteins in a single biological sample. Limitations on the size of the collected sample require that these measurements be done on as small a volume of fluid as possible. This interest has been one of the driving forces behind the development of microfluidic devices for biomedical applications. The move to these smaller-scale systems has a number of advantages. First, they are capable of analyzing smaller volumes of sample. Second, in applications such as capillary electrophoresis, the microfluidic system can achieve the same separation resolution in much less time than a larger-scale system. Finally, the reduced size of the analysis setup raises the possibility of developing portable analytical devices. In collaboration with scientists at NIST, DBEPS is developing a microfluidic device for immunoaffinity electrophoresis, in which multiple proteins will be simultaneously isolated and detected. The device will consist of a microfluidic channel, approximately 25 microns in diameter and a few millimeters in length, in which desired antibodies are bound in a series of localized sections. When the sample fluid flows through the channel, the target proteins will bind to the antibodies. Initially, the proteins will be detected optically, using fluorescent tags. After detection, the captured proteins may be eluted from the channel for further analysis. This device architecture has several advantages. First, the isolation of the proteins is achieved through a single-point capture scheme. Second, the proteins can be recovered from the capillary with their biological activity intact permitting further analysis. Finally, because the proteins can be flushed from the device, the chip can be reused, thus both minimizing the use of expensive antibody libraries and greatly reducing the cost and effort for analysis of a series of samples. Prototype devices have been made at NIST using silicon as the substrate material in which microfluidic channels have been etched to evaluate both bonding methodologies, including anodic bonding, and sealant techniques for sample introduction ports. The immediate clinical need that motivates this project is an epidemiological study of the immune response to Herpes Papilloma Virus (HPV) infection, and the relationship between this response and the development of cervical cancer. Studies of lymphocytes taken from peripheral blood samples have suggested a difference between patients whose cells respond to HPV peptides with a T lymphocyte helper cell type 1 response (Th1-type response) compared to those with a Th-2 type response. In order to better understand these differences at a molecular level, it is necessary to detect and quantify a number of selected immune regulatory molecules directly in cervical secretions, as a local probe, rather than blood samples as a systemic probe of the immune response. From these requirements, the need for a microfluidic device such as the one being developed becomes clear. The volume of cervical secretions that can be collected is quite small, typically 25-50 microliters, and the difficulty in collecting a sufficient number of fluid samples for an epidemiological study makes it imperative to extract as much information as possible from a single sample Conventional diagnostic techniques, such as ELISA, only allow for the analysis of one or two analytes from a sample of this size. The proposed device, will be able to detect over a dozen proteins from a one-microliter sample volume. Although the functionality of the proposed device is being targeted for this particular application, there is every reason to expect that, once this device is realized, many other biological and clinical applications could also be addressed.
