DABA method

Following a 10-fold dilution of the sample with 0.6 M perchloric acid, the product of this reaction absorbs maximally at 420 nm. The original assay, developed by Kissane and Robins,12 uses DABA dihydrochloride for depurination (no perchloric acid was used) and employs fluorescence measurements at 520 nm after excitation at 420 nm. More recent work shows that the assay with perchloric acid can be conducted with 10-pL DNA sample volumes and provides a linear fluorescence calibration curve over the 10-500-ng total DNA range (1-50 pg/mL).13 The DABA method has since been modified for colorimetric measurements at 420 nm, and has a detection limit of 25 pg.14 The DABA reaction is specific for DNA, but... 

Several methods are available for the colorimetric or flourometric determination of total DNA. The first two that will be considered involve depurination of DNA under acidic conditions, and are followed by chemical reactions with 3,5-diamino-benzoic acid (DABA) or diphenylamine (DPA). Two fluorescence methods based on selective, noncovalent interactions of a probe molecule with DNA will then be considered. 

The DPA method is based on the colorimetric measurement of products formed by reaction of co-hydroxylevulinylaldehyde (from the deoxyribose released after depurination of DNA) with diphenylamine in 1 M perchloric acid.14-16 The reaction yields a mixture of products, such as that shown in Eq. 1.4,17 which together have an absorbance maximum at 595 nm. This method has a similar dynamic range to the DABA assay with colorimetric measurement. Interferences include proteins, lipids, saccharides, and RNA. 

After correction for dilution, the ferricyanide method yields 18 3 pg/mL. This concentration of carbohydrate will produce a signal in the DABA assay that is equivalent to 33 pg/mL of DNA (18 x 330/180). The remaining signal in the DABA assay is due to DNA its concentration is therefore 11 pg/mL. A dyebinding assay (e.g., ethidium bromide) can be used as a confirmative method. 

In order to find amphiphilic, less hydrophilic derivatives we used various kinds of organic acids as the source of counter anions of LUC derivatives. Strong acids such as 2-bromoethanesulfonic acid and tetracyanohydroquinone readily formed LUC derivatives, BES and TCHQ in route a. On the other hand, LUC derivatives from rather weak acids such as malonic acid monoethyl ester and benzoic acid could not be purified by conventional methods. Therefore we developed a new synthetic method, route b to afford pure LUC derivatives, MA, MAE, BA, SAL, BrBA, CBA, MMT, FBA, 3,5-DABA. All of them showed characteristic absorption, fluorescence and chemiluminescence spectra of bicridinium di-cation as LUC itself. These features indicate that these derivatives are as useful chemiluminescent probes as LUC. The specific reactivity of MMT toward superoxide among ROS (02, H2O2, HCIO, OH, 02) was examined by using chemically produced ROS. It is confirmed that MMT has the specific reactivity as well as LUC. 

A different method by Aharoni to create two-step rigid polyamide networks employs pendant carboxy groups on the precursor linear chains, instead of nitro or amine. One way of preparing such chains requires the preparation of diacid chloride monomeric species from excess terephthaloyl dichloride and DABA ... 

The linear polyamides with pendant carboxy groups prepared by both methods are reacted with rigid aromatic diamines, such as DABA, in solution under Yamazaki conditions to yield gels of two-step rigid aromatic polyamide networks such as [619] ... 

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