Rapid high throughput assay for fluorimetric detection of doxorubicin--application of nucleic acid-dye bioprobe.
A double stranded DNA based fluorescence bioprobe for anticancer agent (doxorubicin) detection is described. This method provides a new way for sensitive DNA/drug interaction study by a homogeneous assay. The probe employs the long-wavelength intercalating fluorophore TOTO-3 (TT3). The anticancer agent, doxorubicin, which interacts with the DNA-TT3 complex, was indirectly measured by the decrease in the fluorescence intensity. Various oligonucleotides with different sequences were examined. Doxorubicin has preference for the oligonucleotide 5'AGCACG3'. Enhanced fluorescence observed for the TT3 intercalation with this oligonucleotide makes the DNA-dye complex a suitable bioprobe for doxorubicin detection by competitive assay. A home-built CCD camera setup was applied along with 384 well plate assay format for high throughput fluorescence imaging. The detection limit can be as low as 25 ng mL(-1) with an upper limit of 100 microg mL(-1). The recovery test with spiked serum sample shows that this method can be a potential routine method for therapeutic drug monitoring (TDM).
Liu
Yang
Y
Pure and Applied Biochemistry, Lund University, P.O. Box 124, SE-22100 Lund, Sweden.
Danielsson
Bengt
B
D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology*; D015374:Biosensing Techniques; D004247:DNA / Q000737:chemistry; D004317:Doxorubicin / Q000032:analysis* / Q000737:chemistry / Q000494:pharmacology*; D004354:Drug Screening Assays, Antitumor; D004563:Electrochemistry / Q000379:methods; D005456:Fluorescent Dyes / Q000494:pharmacology*; D005470:Fluorometry / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008856:Microscopy, Fluorescence / Q000379:methods; D015335:Molecular Probes / Q000737:chemistry*; D009696:Nucleic Acids / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D012680:Sensitivity and Specificity
D000970:Antineoplastic Agents; D005456:Fluorescent Dyes; D015335:Molecular Probes; D009696:Nucleic Acids; D009841:Oligonucleotides; D004317:Doxorubicin; D004247:DNA