Simple Colorimetric Assay for Quantification of Persistent Free Radicals in Biochars

Persistent free radicals (PFRs) in biochars have been found to possess biotoxicity and cause negative impacts on plant germination and growth; however, the detection of PFRs requires bulky expensive devices that are unsuitable for massive and on-site sample measurements. Herein, we proposed a simple and effective colorimetric method for the quantification of PFRs in biochars. The proposed method was achieved by using tetramethyl benzidine (TMB) as a reporter to recognize PFRs through color development (from colorless to blue). By using the free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) as a standard, the absorbance of the color-developed TMB was transformed into the concentration of PFRs. With the developed method, the PFR concentrations in 15 biochars derived from different biomasses at different pyrolysis temperatures were determined and significantly correlated with those determined by the electron paramagnetic resonance method (Pearson's r of 0.960; P < 0.01). The high-throughput detection application was tested by employing a commercial smartphone to take photographs of color-developed TMB and acquire their color signal. The results demonstrated that the proposed method can be used to quantify PFRs in biochars and consequently has applications in the fields of environmental remediation and soil conditioning.