IEEE SENSORS JOURNAL, VOL. 9, NO. 12, DECEMBER 2009 1697 Label-Free Photonic Crystal Biosensor Integrated Microﬂuidic Chip for Determination of Kinetic Reaction Rate Constants Charles J. Choi, Ian D. Block, Brian Bole, David Dralle, and Brian T. Cunningham, Senior Member, IEEE Abstract—We demonstrate a photonic crystal integrated mi- croﬂuidic chip that is compatible with a 384-well microplate format for measuring kinetic reaction rate constants in high-throughput biomolecular interaction screening applications. The device en- ables low volume kinetic analysis of protein–protein interactions with low ﬂow latency, and control of ﬁve analyte ﬂow channels with a single control point. The structure is fabricated with a replica molding process that produces the submicron photonic crystal structure simultaneously with the micrometer-scale ﬂuid channel structure. The device signiﬁcantly reduces the kinetic assay time required compared with a conventional biosensor microplate in which reagents reach the active detection surface by diffusion. Using the photonic crystal sensor ﬂuid network system, we demon- strate determination of the kinetic association/dissociation rate constants between immobilized ligands and analytes in the ﬂow stream, using the heparin/lactoferrin system as an example. Index Terms—Biomedical transducers, ﬂexible structures, op- tical resonance. I. INTRODUCTION T HE ABILITY to perform biochemical and cellular anal- ysis using small reagent volumes and high measurement throughput has been one of the driving forces behind the development of microﬂuidic lab-on-a-chip (LOC) devices and micro-total-analysis systems [1]–[3]. Often, such systems are produced using microfabrication methods upon glass or silicon substrates with custom-designed interfaces that allow microliter quantities of reagents to be introduced into a system of microﬂuidic channels. However, within the ﬁeld of pharmaceutical discovery and laboratory-based diagnostic assays, a great deal of liquid handling infrastructure currently exists for interfacing with standard 96, 384, and (more recently) 1536-well microplates. For this reason, it is desirable for a label-free biosensing system to easily integrate with these standard formats to enable high throughput in a single-use Manuscript received May 07, 2009; accepted August 06, 2009. Current ver- sion published October 21, 2009. This work was supported in part by the Na- tional Science Foundation under Award DMI 0328162 and 0427657. Any opin- ions, ﬁndings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reﬂect the views of the Na- tional Science Foundation. This work was also supported by SRU Biosystems. The associate editor coordinating the review of this paper and approving it for publication was Dr. M. Abedin. The authors are with the University of Illinois at Urbana–Champaign, Urbana, IL 61801 USA (e-mail: cjchoi@illinois.edu; iblock2@illinois.edu; bcunning@illinois.edu). Color versions of one or more of the ﬁgures in this paper are available online at http://ieeexplore.ieee.org. Digital Object Identiﬁer 10.1109/JSEN.2009.2030666 disposable format. This requirement has driven the commercial adoption of photonic crystal (PC) biosensor microplates for applications in pharmaceutical high-throughput screening for measuring protein–protein interactions [4]–[7], protein-small molecule interactions [8], cell-based assays [9], [10], and cell-drug interactions [11]. While label-free optical biosensors embedded within the bottom surface of microplate wells offer a convenient high-throughput detection system, the kinetics that drive detection of biomolecules to attach to the sensor surface is based mainly upon diffusion. Many publications have demonstrated the efﬁcacy of biosensors interfaced with mi- croﬂuidic channels as a means for obtaining detection kinetics that are limited by chemical reaction rates. These can serve as a rapid and sensitive means for characterizing ligand-analyte binding afﬁnity constants through the rate of change of detected biosensor signal [12]–[16]. Recently, we demonstrated the co-fabrication of PC biosen- sors with a network of microﬂuidic channels in which a single nanoreplica molding step from a silicon “master” template wafer that contains the micrometer-scale surface structure for microﬂuidic channels and the nanometer-scale surface features for the PC biosensor structure. The resulting PC sensors and ﬂuid channels were automatically self-aligned, and were fab- ricated over a 3 5 inch area on ﬂexible plastic substrates for integration with a standard 96-well microplate. We also demon- strated a simple valveless control scheme in which some wells are designated as “control” wells for driving the introduction of immobilized ligands and detected analytes through microﬂu- idic channels for real-time monitoring of up to 11 biochemical binding interactions in parallel with a high-resolution label-free imaging detection instrument [17]. With the device, reduction in the endpoint binding assay time was achieved, but kinetic analysis could not be effectively performed with the use of a long ﬂow channel length (64 mm), which was required to bring analytes from the “analyte” microplate wells to the central measurement point. To ensure equal ﬂow rate for a pneumatic pressure applied equally to all “analyte” wells, serpentine ﬂow paths were implemented for wells with closest proximity to the measurement point. Despite these efforts, the previously reported chip exhibited ﬂow rate differences between analyte ﬂow channels, and limitations on the maximum achievable ﬂuid ﬂow rate. In this paper, we demonstrate PC biosensor integrated mi- croﬂuidic channels compatible with a 384-well microplate format. The device structure reported here enables low volume kinetic analysis of protein–protein interactions through ﬁve analyte ﬂow channels with a single control point and offers 1530-437X/$26.00 © 2009 IEEE Authorized licensed use limited to: University of Illinois. Downloaded on January 8, 2010 at 16:07 from IEEE Xplore. Restrictions apply.