Development of the ultra-weak chemiluminescence method based on luminol reaction for use in the detection of ultra-trace levels of blood

In this study, we focused on ultra-weak chemiluminescence (uwCL) measurements and aimed to develop a blood detection method at trace levels, that are difficult to observe using a conventional luminol reaction method (visual observation). Furthermore, we investigated sampling methods that could detect trace bloodstains from the field in our laboratory settings. To achieve these highly sensitive detection levels in the uwCL measurements, the optimal measurement conditions were established as follows: the luminol reaction solution chosen was a mixture of 6.0 × 10 −3 % luminol/1.5 × 10 −2 % sodium hydroxide solution and 1.5 × 10 −2 % hydrogen peroxide water (6:1); the temperature in the sample chamber was set to 20 °C; the sample chamber was filled with an oxygen displacement atmosphere; the sample chamber was filled with 3 mL of 0.01% sodium hydroxide solution prior to the experiment, and the measurement wavelength was set to 460 nm. Using the developed method, blood diluted to 12.5 million-fold (8.0 ng equivalent of the absolute weight of whole blood) was detectable, and high linearity ( r = 0.9986) between uwCL intensity and whole blood equivalent was observed in the range of 8.0–100 ng. In contrast, the detection limit of the conventional method was 1.0 µg of the whole blood equivalent. Thus, the uwCL method was approximately 125 times more sensitive than the conventional method. In addition, we demonstrated that the sampling method of wiping with a melamine sponge soaked in a 10% sodium dodecyl sulfate solution is effective for sampling evidence materials at an appraisal site suspected of having traces of adhered blood. Graphical abstract