Anthracycline-Functionalized Dextran as a New Signal Multiplication Tagging Approach for Immunoassay

Abstract

The most used kind of immunoassay is enzyme-linked immunosorbent assay (ELISA); however, enzymes suffer from steric effects, low stability, and high cost. Our research group has been developing quinone-linked immunosorbent assay (QuLISA) as a new promising approach for stable and cost-efficient immunoassay. However, the developed QuLISA suffered from low water-solubility of synthesized quinone labels and their moderate sensitivity. Herein, we developed a new approach for signal multiplication of QuLISA utilizing the water-soluble quinone anthracycline, doxorubicin, coupled with dextran for signal multiplication. A new compound, Biotin-DexDox, was prepared in which doxorubicin was assembled on oxidized dextran 40, and then it was biotinylated. The redox-cycle-based chemiluminescence and the colorimetric reaction of Biotin-DexDox were optimized and evaluated, and they showed very good sensitivity down to 0.25 and 0.23 nM, respectively. Then, Biotin-DexDox was employed for the detection of biotinylated antibodies utilizing avidin as a binder and a colorimetric assay of the formed complex through its contained doxorubicin redox reaction with NaBH4 and imidazolium salt yielding strong absorbance at 510 nm. The method could detect the plate-fixed antibody down to 0.55 nM. Hence, the application of Biotin-DexDox in QuLISA was successfully demonstrated and showed a significant improvement in its sensitivity and applicability to aqueous assays.

Biotinylated Quinone as a Chemiluminescence Sensor for Biotin-Avidin Interaction and Biotin Detection Application

“…In some reports, the targeted biotinylated compound has an absorbance that overlaps with the HABA assay measurement wavelength. In such cases, it is impossible to detect because of the interference encountered [22]. On the other hand, our method is simple and easily undertaken with a simple instrument (luminometer), with no need to use any extra instruments.…”

“…Additionally, quinones were used as a new alternative for replacing enzymes in enzyme-linked immunosorbent assay (ELISA), as our research groups have developed quinone-linked immunosorbent assay (QuLISA) [20] and multi-QuLISA [21,22].…”

“…In a solution containing biotin, BT-NQ, and avidin, there will be a competition between the free biotin and BT-NQ for the interaction with avidin. This competition was used here to make a competitive assay for biotin determination similar to the HABA reagent assay (4 -hydroxyazobeneze-2-carboxylic acid), which is essentially a displacement test [21,22,24]. At first, the free biotin displaces the BT-NQ from its interaction with avidin because pristine biotin (in this case, the free biotin) has a higher affinity to avidin than the modified biotin (BT-NQ).…”

Optical Bioassays Based on the Signal Amplification of Redox Cycling

“…The produced superoxide anions could react with luminal molecules to generate a long-lived and strong chemiluminescence signal. Thanks to the interesting redox cycling, different quinone derivatives or quinoneloaded nanomaterials have been used and developed as the labels for the development of enzyme-free biosensors [105]. For example, El-Maghrabey et al used 2-(9-carboxynonyl)-1,4-naphthoquinone (NQ) as the signal-generating tag to modify biotin (BT) and then developed a chemiluminescence method for avidin detection [106].…”

“…Under the optimized conditions, the food allergen, β-casein, was determined within a dynamic range of 78.1-2500 ng/mL with a detection limit of 3 ng/mL. Furthermore, dextran was utilized by Kuroda's group to load biotin and water-soluble quinine anthracycline (doxorubicin) for signal multiplication [109]. Electrochemiluminescence is a kind of luminescence that can emit from a molecular excited species formed at or near the electrode surface via electrochemical reactions [110].…”