Field-flow fractionation of cells with chemiluminescence detection.
Field-flow fractionation is a separation technique characterized by a retention mechanism which makes it suitable for sorting cells over a short analysis time, with low sample carry-over and preserving cell viability. Thanks to its high sensitivity, chemiluminescence detection is suitable for the quantification of just a few cells expressing chemiluminescence or bioluminescence. In this work, different formats for coupling gravitational field-flow fractionation and chemiluminescence detection are explored to achieve ultra-sensitive cell detection in the framework of cell sorting. The study is carried out using human red blood cells as model sample. The best performance is obtained with the on-line coupling format, performed in post-column flow-injection mode. Red cells are isolated from diluted whole human blood in just a few minutes and detected using the liquid phase chemiluminescent reaction of luminol catalysed by the red blood cell heme. The limit of detection is a few hundred injected cells. This is lower than the limit of detection usually achieved by means of conventional colorimetric/turbidimetric methods, and it corresponds to a red blood cell concentration in the injected sample of five orders of magnitude lower than in whole blood.
Melucci
Dora
D
Department of Chemistry G. Ciamician, University of Bologna, Via Selmi 2, I-40126 Bologna, Italy. dora.melucci@unibo.it
Roda
Barbara
B
Zattoni
Andrea
A
Casolari
Sonia
S
Reschiglian
Pierluigi
P
Roda
Aldo
A
D004912:Erythrocytes / Q000166:cytology*; D031521:Fractionation, Field Flow; D006418:Heme / Q000737:chemistry; D006801:Humans; D049449:Luminescence; D008165:Luminol / Q000737:chemistry; D012680:Sensitivity and Specificity
D006418:Heme; D008165:Luminol