Anal. Chem. 2009, 81, 4043–4047
Three-Minute-Long Chemiluminescent Immunoassay Using Dually Accelerated Immunoreaction by Infrared Heating and Passive Mixing Hong Liu,† Zhanjun Yang,† Feng Yan,*,‡ Yueming Xu,† and Huangxian Ju*,† Key Laboratory of Analytical Chemistry for Life Science, Ministry of Education of China, Department of Chemistry, Nanjing University, Nanjing 210093, People’s Republic of China, and Jiangsu Institute of Cancer Research, Nanjing 210009, People’s Republic of China A rapid chemiluminescent (CL) immunoassay method was developed by integrating a newly designed infraredradiation technique with a pressure-driven fluidic system. The fluidic system combined a three-dimensional helical glass tube for rapid mixing of immunoreagents with two spiral glass tubes for magnetic separation and CL detection, respectively. The mixture passively formed in the helical glass tube could be quickly heated and kept at about 37 °C by the infrared radiation. The immunoreaction could be finished within 90 s due to the dual acceleration by the improved mass transport and enhanced reaction kinetics. The horseradish peroxidaselabeled sandwich immunocomplex formed on paramagnetic particles was then separated by a permanent magnet and mixed with CL substrate in a long spiral tube, and the detection mixture was immediately injected through another spiral tube in front of the photomultiplier for collecting the CL signal. Using r-fetoprotein as a proofof-principle analyte, the immunoassay could be completed within 3 min with a linear calibration range of 0.2-90 µg/L. This programmable method showed acceptable detection and fabrication reproducibility and good accuracy, indicating a promise of automated high-throughput clinical application. Automated immunoassay has recently attracted considerable interest due to its advantages in reducing cost, labor requirement, and turnaround time,1 and improved performance.2 The automation can be achieved by combining a flow injection system with microarray,3 surface-renewable immunosensors,4,5 paramagnetic microbeads (PMs),6-8 and polymer beads.9 The PMs have extensively been used for development of fluidic immunoassay methods due to their large surface areas and easy handling as suspension and capture by magnet. However, automated immunoassay based on PMs in flow injection systems still poses several * To whom correspondence should be addressed. Phone/Fax: +86-2583593593. E-mail:
[email protected] (F.Y.);
[email protected] (H.J.). † Nanjing University. ‡ Jiangsu Institute of Cancer Research. (1) Lori, J. S.; Daniel, W. C. Anal. Chem. 1999, 71, 356R–362R. (2) Ahn, K. C.; Lohstroh, P.; Gee, S. J.; Gee, N. A.; Lasley, B.; Hammock, B. D. Anal. Chem. 2007, 79, 8883–8890. 10.1021/ac900245x CCC: $40.75 2009 American Chemical Society Published on Web 03/26/2009
problems: (1) at low flow rate, for instance, 100 µL/s (0.13 m/s), at which the Reynolds number is 126, much lower than the threshold value of 2000, flows in typical channel (cross-sectional dimension,
