# Carbon Nano-Onion Peroxidase Composite Biosensor for Electrochemical Detection of 2,4-D and 2,4,5-T
## 1 Introduction  
Carbon nanomaterials emerged as one of the most suitable materials in biosensing due to a combination of chemical versatility and electronic properties [1]. In particular, carbon nano-onions (CNOs) were recently revealed as suitable electrode materials for the detection of small molecules [2, 3], antibodies [4] and glyphosate [5]. CNOs are formed by concentric graphitic layers with increasing diameters and possess a distinctive combination of high conductivity and biocompatibility, among other interesting properties [6]. They can be easily modified by a wide range of functional groups and incorporated in composite materials with organic or inorganic polymers for a wide range of applications, including energy storage [7] and biomedicine [8], among others.  
Phenoxy herbicides, such as 2,4-dichlorophenoxyoxyacetic acid (2,4-D) and 2,4,5-trichlorophenoxyoxyacetic acid (2,4,5-T), are synthetic analogs of auxin plant growth hormones derived from phenoxyacetic acid [9]. They were successfully used to control broad-leaved weeks in several crops, as well as in forestry, urban parks and domestic gardens but are also considered a source of environmental contamination of soil and groundwater with potential carcinogenic effects in humans [10]. Hence, sensitive detection methods were developed for monitoring these herbicides. These include, but are not limited to, chromatography and immunoassay, which often need to be combined with extraction methods to guarantee their isolation and enrichment in complex samples such as soil [11].  
Enzymatic biosensors based on enzyme activity inhibition were developed for pesticide detection as an alternative to traditional methods due to their simplicity and reusability [12, 13]. There are only a few reports on the use of this strategy to detect phenoxy herbicides. In an early work, the inhibitory effect of 2,4-D and 2,4,5-T on the activity ofcholinesteraseterase was used to develop an amperometric biosensor based on the use of antibodies in solution or immobilized in nitrocellulose membranes [14]. Alkaline phosphatase (ALP) immobilized on sol-gel/chitosan film on carbon paste electrodes was used to detect these herbicides in the presence of heavy metals based on the inhibition of ascorbic acid 2-phosphate hydrolysis [15]. More recently, [PERSON] et al. reported the inhibition mechanism of 2,4-D to catalase supported on porous calcium phosphate and the construction of a biosensor based on a flow-injection analysis system to detect 2,4-D in commercial bean sprouts samples [16].  
Horseradish peroxidase (HRP, EC 1.11.1.7) is a heme-containing metalloenzyme that utilizes peroxides such as hydrogen peroxide to oxidize a wide variety of organic and inorganic compounds. It is perhaps the most used enzyme in bioanalysis due to its high stability, ease of conjugation, low price compared to other widespread alternatives such as ALP and high turnover rate that allows the generation of strong signals in a relatively short time [17]. Its inhibition was only applied to detect glyphosate and its metabolites [18; 19] with very little application to other herbicides [20].  
Herein we report the inhibition mechanism of 2,4-D and 2,4,5-T on HRP activity and the development of an amperometric biosensor for the detection of these herbicides in soil and river water samples. HRP was conjugated to activated CNOs and immobilized on the surface of screen-printed carbon electrodes (SPE) using a \(\beta\)-cyclodextrintrim polymer as a cross-linker. Detection is based on the inhibition of HRP activity, which, to the best of our knowledge, has not been used for the detection of this family of herbicides. The effect of the CNOs on the analytical parameters and biosensor stability was also studied.