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

Biotin, or vitamin B7, is essential for metabolic reactions. It must be obtained from external sources such as food and biotin/vitamin supplements because it is not biosynthesized by mammals. Therefore, there is a need to monitor its levels in supplements. However, biotin detection methods, which include chromatographic, immune, enzymatic, and microbial assays, are tedious, time-consuming, and expensive. Thus, we synthesized a product called biotin-naphthoquinone, which produces chemiluminescence upon its redox cycle reaction with dithiothreitol and luminol; then it was used as a chemiluminescence sensor for biotin-avidin interaction. When a quinone biotinylated compound binds avidin, the chemiluminescence decreases noticeably due to the proximity between quinone and avidin, and when free biotin is added in a competitive assay, the chemiluminescence returns. The chemiluminescence is regained as the free biotin displaces biotinylated quinone in its complex with avidin, freeing biotin-naphthoquinone. Many experiments, including the use of a biotin-free quinone, proved the competitive nature of the assay. The competitive assay method used in this study was linear in the range of 1.0-100 µM with a detection limit of 0.58 µM. The competitive chemiluminescence assay could detect biotin in vitamin B7 tablets with good recovery of 91.3 to 110% and respectable precision (RSD < 8.7%).

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

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.

Development of signal multiplication system for quinone linked immunosorbent assay (Multi-QuLISA) by using poly-l-lysine dendrigraft and 1,2-naphthoquinone-4-sulfonate as enzyme-free tag.

A sensitive and stable signal multiplied quinone-linked immunosorbent assay (Multi-QuLISA) was developed. In Multi-QuLISA, an oligomerized quinone linked to biotin, namely biotin-8mer-naphthoquinone (Bio8mer-NQ), is used as a signal-generating label. Bio8mer-NQ is formed from a dendrigraft poly-l-lysine generation 1 (DPLL G1), a controlled branched oligomer composed of eight lysine moieties with nine free amino groups as a backbone. One of the nine amino groups of DPLL G1 is attached to biotin moiety, while the other eight are attached to 1,2-naphthoquinone-4-sulfonate (NQS). Bio8mer-NQ labels a biotinylated detection antibody using avidin as a co-binder. Then, multi-quinones in Bio8mer-NQ undergo a redox cycle with dithiothreitol and luminol, generating strong chemiluminescence. Standard ELISA uses a label enzyme that suffers from vulnerability in different conditions and poor stability. Bio8mer-NQ showed better stability than the enzyme (biotin-HRP) under different drastic pH and temperature conditions, hydrolytic enzymes, etc. Furthermore, Bio8mer-NQ was used as both chemiluminescence and colorimetric label based on the redox cycle of quinone, and it had LODs of 1.5 and 6.5 nM, respectively. The method could detect biotinylated immunocomplex in an in-house designed immunoassay down to 0.2 nM, which is about 25 times more sensitive than biotin HRP. Eventually, Bio8mer-NQ was applied successfully in Multi-QuLISA for detecting ß-casein with a sensitivity of 3.2 ng/mL, while the conventional ELISA had an LOD of 35 ng/mL. Overall, Bio8mer-NQ is a stable compound that could be used as an excellent replacement for the enzyme in immunoassay and can be used in both colorimetric and chemiluminescence assays with good sensitivity.