Design and manufacture of capillary wicks for ultrasensitive detection of antigenic and nucleic acid analytes

Design and manufacture ofcapillary wicks for ulirasensitive detection ofantigenic and nucleic acid analytesNaheed A. Mufti, Troy Tagg, Rob Webb, and Luke V. SchneiderSRI International333 Ravenswood AvenueMenlo Park, CA 94025ABSTRACTUsing upconverting phosphor reporter probes to detect antigenic and nucleic acid analytes in environmental samples requiredthe design and manufacture of a novel sampling and assay device that exploits the unique characteristics of the upconvertingphosphors. The absence of natural materials that upconvert energy make single particle detection possible with thesereporters. Ultrasensitive detection and quantitation of analytes is governed by the area of the capture surface, the density ofthe capture probes, as well as the specific upconversion efficiency of the phosphor label. The optimization of a flow channeland capture surface for single phosphor particle detection is a complex function ofthe volume ofsample that is drawn throughthe channel, the probability that this sample volume contains an analyte particle, the fraction of analytes contained in thesampled volume that bind to the capture site, and the optical dimensions of the capture site. The result ofthis optimizationusing theoretical diffusion models for submicron upconverting phosphor particles was a rectangular flow channel with a 200 x300 im capture surface. Capture surfaces ofthis size have been prepared using photo-directed synthesis methods inside glasscapillary wicks that have been subsequently used for the phosphor-based assays.Keywords: Upconverting phosphors, in vitro diagnostics, capillary wicks1. INTRODUCTIONUpconverting phosphors are inexpensive, inorganic, crystalline materials that provide an almost immediate response(millisecond time scale) once excited by JR light ofthe proper wavelength. Furthermore, unlike fluorescent dyes, they do notphotobleach. Upconverting phosphors have the unique property of absorbing 2 or 3 photons oflow energy (IR) light to emita single higher energy (visible) photon. This unique property enables high sensitivity detection in diagnostic assays.1Because the upconversion process is unique, there is no background phosphorescence from the carrier fluid or the assaybiochemistry. Assay avidity such as nonspecific adsorption of the phosphors to the capture site is the only background ofconcern, and this can be controlled with appropriate blocking chemistries as in other immuno-diagnostic (ID) assays.Because upconversion occurs within the host crystal, the optical properties ofthe phosphors are completely unaffected by theirenvironment (e.g., buffers and changes in assay temperature). Consequently, the detection process is unaffected by thesampled fluid and is robust with respect to the field sampling conditions.Multiple spectrally unique phosphor colors exist. If each ofthese spectrally unique colors is attached to a different detectionprobe (i.e., antibody or nucleic acid oligomer), then simultaneous multiplexing of diagnostic assays is possible. Spectraldiscrimination and quantitation ofthe unique optical signature ofeach phosphor is easily accomplished because ofthe narrowemission bands. Furthermore, as shown in Figure 1, multiple spectrally unique emissions can be obtained with differentphosphors all excited by the same source, a 980 nm diode laser. Because the emission wavelengths (400 -