Plastic ELISA-on-a-chip based on sequential cross-flow chromatography.
A plastic chip that can perform immunoassays using an enzyme as signal generator, i.e., ELISA-on-a-chip, was developed by incorporating an immunostrip into channels etched on the surfaces of the chip. To utilize an analytical concept of cross-flow chromatography, the chip consisted of two cross-flow channels in the horizontal and vertical directions. In the vertical channel, we placed a 2-mm-wide immunostrip for cardiac troponin I (cTnI), which was identical to a conventional rapid test kit except for the utilization of an enzyme, horseradish peroxidase (HRP), as tracer. An enzyme substrate supply channel and a horizontal flow absorption pad compartment were transversely arranged on each lateral side of the signal generation pad of the strip, respectively. Upon application of a sample containing cTnI, it migrated vertically through the membrane strip by capillary action, and antigen-antibody binding occurred. After 15 min, the horizontal flow was initiated by the addition of a chromogenic substrate solution for HRP into the supply channel and by partial superimposition of the horizontal flow absorption pad onto the signal generation pad. A color signal proportional to the analyte concentration was produced on this pad, measured after 5 min as optical densities using a digital camera-based detector, and quantified by integration of the densities under the peak after normalization. Its calibration curve indicated that the detection limit of the chip was approximately 0.1 ng/mL and its quantification limit was 0.25 ng/mL. In measuring blindly prepared samples, the chip performance correlated with that of a reference system, Beckman Coulter Access, within 2.5-fold discrepancy at the detection limit.
Cho
Joung-Hwan
JH
Graduate School of Biotechnology, Korea University, Sungbuk-ku, Seoul.
Han
Seung-Mok
SM
Paek
Eui-Hwan
EH
Cho
Il-Hoon
IH
Paek
Se-Hwan
SH
D000906:Antibodies / Q000737:chemistry; D000915:Antibody Affinity; D002845:Chromatography / Q000379:methods*; D003124:Colorimetry; D004797:Enzyme-Linked Immunosorbent Assay / Q000295:instrumentation / Q000379:methods; D006735:Horseradish Peroxidase / Q000737:chemistry; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods*; D040081:Protein Array Analysis / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors; D019210:Troponin I / Q000032:analysis*
D000906:Antibodies; D019210:Troponin I; D006735:Horseradish Peroxidase