Optical scanner for immunoassays with up-converting phosphorescent labels.
A 2-D optical scanner was developed for the imaging and quantification of up-converting phosphor (UCP) labels in immunoassays. With resolution better than 500 microm, a scan rate of 0.4 mm/s, and a 1-2% coefficient of variation for repeatability, this scanner achieved a detection limit of fewer than 100 UCP particles in an 8.8. x 10(4) microm(2) area and a dynamic range that covered more than three orders of magnitude. Utilizing this scanner, a microfluidic chip immunoassay for the cytokine interferon-gamma (IFN-gamma) was developed: concentrations as low as 3 pM (50 pg/mL) were detected from 100 microL samples with a total assay time of under an hour, including the 8 min readout. For this UCP-based assay, 2-D images of the capture antibody lines were scanned, image processing techniques were employed to extract the UCP emission signals, a response curve that spanned 3-600 pM IFN-gamma was generated, and a five-parameter logistic mathematical model was fitted to the data for determination of unknown IFN-gamma concentrations. Relative to common single-point or 1-D scanning optical measurements, our results suggest that a simple 2-D imaging system can speed assay development, reduce errors, and improve accuracy by characterizing the spatial distribution and uniformity of surface-captured optical labels as a function of assay conditions and device parameters.
Li
Janice J
JJ
Department of Electrical Engineering, Stanford University, Stanford, CA 94305, USA. janice.li@sri.com
Ouellette
Amy L
AL
Giovangrandi
Laurent
L
Cooper
David E
DE
Ricco
Antonio J
AJ
Kovacs
Gregory T A
GT
D004867:Equipment Design; D019544:Equipment Failure Analysis; D007118:Immunoassay / Q000295:instrumentation* / Q000379:methods; D007834:Lasers; D008163:Luminescent Measurements / Q000295:instrumentation* / Q000379:methods; D055095:Optics and Photonics / Q000295:instrumentation*; D013194:Staining and Labeling / Q000379:methods