Lateral-flow and up-converting phosphor reporters to detect single-stranded nucleic acids in a sandwich-hybridization assay.
Up-converting Phosphor Technology (UPT) particles were used as reporters in lateral-flow (LF) assays to detect single-stranded nucleic acids. The 400-nm phosphor particles exhibit strong visible luminescence upon excitation with infrared (IR) light resulting in the total absence of background autofluorescence from other biological compounds. A sandwich-type hybridization assay was applied using two sequence-specific oligonucleotides. One of the oligonucleotides probes was covalently bound to the UPT particle (reporter) for direct labeling and detection, whereas the second oligonucleotide probe contained biotin for capture by avidin during LF. The whole procedure of hybridization, UPT-LF detection, and analysis required a minimum time of 20 min. Moreover, aiming at minimal equipment demands, the hybridization conditions were chosen such that the entire assay could be performed at ambient temperature. During lateral flow, only targets hybridized to both capture and detection oligonucleotide were trapped and detected at an avidin capture line on the LF strip. Analysis (IR scanning) of the strips was performed in an adapted microtiter plate reader provided with a 980-nm IR laser for excitation of the phosphor particles (a portable reader was also available). Visible luminescence was measured and presented as relative fluorescence units (RFU) allowing convenient quantitation of the phosphor signal. With the assay described here as little as 0.1 fmol of a specific single-stranded nucleic acid target was detected in a background of 10 microg fish sperm DNA.
Corstjens
Paul L A M
PL
Laboratory for Cytochemistry and Cytometry, Department of Molecular Cell Biology, Leiden University Medical Center, Wassenaarseweg 72, The Netherlands. Corstjens@LUMC.NL
Zuiderwijk
Michel
M
Nilsson
Mats
M
Feindt
Hans
H
Sam Niedbala
R
R
Tanke
Hans J
HJ
D000818:Animals; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000032:analysis* / Q000235:genetics; D005399:Fishes / Q000235:genetics; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization / Q000379:methods*; D015345:Oligonucleotide Probes / Q000032:analysis* / Q000235:genetics; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature
D004277:DNA, Single-Stranded; D015345:Oligonucleotide Probes