Organophosphates, organophosphonates (henceforth jointly abbreviated as OP) and carbamates are typical anticholinergic compounds which are widely known as pesticides or nerve agents [1]. Their common toxicological pathway is based on multiple mechanisms, including increased oxidative stress and modification of blood proteins; however, their main mechanism of action is esterification of active serine in serine hydrolases [2]. Especially, blood acetylcholinesterase (AChE; EC 3.1.1.7), butyrylcholinesterase (BChE; EC 3.1.1.8) and (β-glucuronidase; EC 3.2.1.31) are typical markers of intoxication [3]. The most important toxicology pathway of OP as well as carbamates is based on inhibition of AChE in neurosynapses. AChE terminates neurotransmission through neurosynaptic cleft in both central and peripheral nervous system; accumulated acetylcholine may overstimulate nicotinic and/or muscarinic receptors [4]. Symptomatic manifestation of intoxication typically consists from bronchospasms, bradycardia, miosis, lacrymation, diarrhoea, salivation; moreover, typical symptoms of CNS nicotinic and muscarinic receptors overstimulation would also occur: confusion, coma, agitation and/or respiratory failure [5].

The sensitivity of cholinesterases to the inhibitory effects of OP, carbamates and some natural toxins such as aflatoxins and drugs suitable for treatment of symptomatic manifestations of Alzheimer’s disease was found to be applicable to the construction of biosensors for the assay of the given inhibitors [6–8,34]. Though the principle of the assay is the same, OPs, in contrast to Alzheimer’s disease drugs and aflatoxins, are irreversible inhibitors. Many methods are widely available for the construction of cholinesterase based biosensors (CBBs). Amperometric [9–10], potentiometric [11–13] and optical, including non-linear optics [14–16], biosensors are typical examples of CBBs. Voltammetric techniques are applicable for the assay of multiple analytes [17–19]. The common voltammetric principle of cholinesterase activity measurement is typically based on electrochemical oxidation of nascent thiocholine coming from acetylthiocholine [20,21]. Presence of OP in analyzed sample leads to the stopping of the mentioned reactions.

The sffects of ionizing radiation on cholinesterase activity in vivo has not been widely studied. Krokosz et al. proposed that that total activity of AChE in human erythrocytes would change due to X-rays [22]; moreover, Dimberg et al. found an increase of AChE activity, as well as nerve growth factor protein in mice brain after exposure to X-rays [23]. The mentioned studies on in vivo impact on the body and changes of AChE activity would be on a transcription level. The presented study is aimed at following of changes of intercepted AChE on an electrochemical strip during exposition to radiation. Durability of biosensors under radiation and the impact on analytical parameters is considered. Moreover, prediction of achieved results to a viable body will be made.

Biosensors were constructed as described in the Experimental section. Thirty five freshly prepared biosensors were separated into seven groups. Another ten strips were used in experiments without immobilization of AChE or any other modification. Biosensors were kept under standard laboratory conditions and all radiation as well as measurements were carried out under these conditions.

Twelve groups (n = 5) of biosensors were irradiated with doses of 5 mGy to 100 kGy. Two groups were kept as a control. Two groups of biosensors were prepared for one radiation dose. The first group was used to investigate the activity of AChE in the absence of paraoxon, while the second group was used to investigate the AChE activity in the presence of paraoxon. Data obtained are summarized in Figure 1.

We expected a decrease of immobilized enzyme activity as the ionizing radiation exceeded the typical mortal dose. We suggest that the lethal dose of radiation is individual and strongly depends on the time of exposure. A dose of 1 Gy within one hour causes radiation sickness, vomiting, diarrhea, and hemorrhage. Incidence of cancer would be abrupt in the future. Doses of 2–5 Gy lead to acute symptoms and extensive mortality. However, no significant differences in the control biosensors were found, even when biosensors were extensive irradiated. Measured current fluctuated in a range from 395 to 455 nA. The values were overlaid within their standard deviations and no difference or correlation to radiation dose was found. The fact would be surprising when the typical effects on the body are considered [24]. The data indicate good stability of AChE when exposed to ionizing radiation and wide stability of immobilized AChE would be also expected [25]. The overall stability of biosensors was previously estimated [10].

Though the immobilized AChE displayed no specific changes in activity after exposure to ionizing radiation, a surprising result was achieved when paraoxon was assayed. A 1 ppm solution of paraoxon was assayed by biosensors previously exposed to radiation as well as the control ones. Residual activity of AChE of around 22% (current 94 ± 15 nA) remained when the activity of the control biosensors was considered. A quite different phenomenon arose when the irradiated biosensors were used for assay purposes. The residual current provided by irradiated biosensors was slightly lower when the dose of radiation was lower than 50 Gy. However, the decrease was not significant. The current provided by biosensors consequently exposed to radiation and paraoxon was 79 ± 9 nA. On the other hand, doses of 50 Gy – 100 kGy potentiated AChE to be extensively inhibited by paraoxon. The lowest current (the most extensive inhibition) was found at biosensors irradiated by 5 Gy. It was 49 ± 6 nA, representing a residual activity of 11%. In order to find out whether the residual activity would decrease more, biosensors irradiated by 100 kGy were used. The achieved current after inhibition by paraoxon was 55 ± 10 nA. It seems that radiation above 5 kGy has no further effect on the sensitivity of AChE-based biosensors towards the representative inhibitor paraoxon. We should imention that the dose of 100 kGy is quite high. There were e.g., found changes in the color of the ceramic support of the electrochemical strip and overall blackening of the plastic holder used in the experiments.

As described above, an increased sensitivity of AChE to the inhibitory effects of paraoxon was found in vitro using electrochemical biosensors. It should be clearly stated that the effect would be also expected to occur in vivo when a viable body is simultaneously exposed to ionizing radiation and an organophosphate. In a wide context, this fact could be also contributing to the commonly known cumulative health risks [26]. Confirmation of the health effect of simultaneous exposure may be expected in the future.

Since ionizing radiation was found to be able to influence the sensitivity of AChE to inhibition by the organophosphate paraoxon, calibration was considered for the subsequent experiments. As mentioned above, deterioration of the AChE-based biosensors due to ionizing radiation was not observed. To the contrary, an improvement of analytical parameters would be expected due to the increased sensitivity towards the inhibitory effect of the organophosphate paraoxon.

Calibration curves for the biosensors are presented in Figure 2. Two calibrations were performed. The first curve presented in the figure was achieved for the performance of the biosensor without any stimulation by radiation. The second curve was obtained when paraoxon was assayed with a biosensor irradiated by a 5 kGy radiation dose. The calibration curves were found to differ; however, the difference was gradual as the concentration of paraoxon increased. The limit of detection was calculated as a point on calibration curve responding to triple de error of the control (no paraoxon) i.e., S/N = 3. The lowest limit of detection for biosensors without stimulation by radiation was 3.8 ppb. The irradiated biosensor provided a lower limit of detection when compared with the non-irradiated ones: 2.5 ppb. The effect of radiation appears to be most effective in the range of higher paraoxon concentration. On the other hand, a 49% shift of the limit of detection was found. This correlated appropriately with data presented in Figure 1.

The above mentioned calibrations point to improved calibration curves provided by biosensors challenged by ionizing radiation. It also represents a possible route in the continuous effort to improve assays where AChE is a promising recognition element [27]. It seems that pre-activation of biosensors would be an appropriate way to improve available biosensors.

The phenomenon that sensitivity of AChE to organophosphates inhibition would change after exposition to ionizing radiation is quite a surprising fact. No relevant studies are currently available in scientific databases. Both the substrate and inhibitor were applied after radiation therefore an impact of these molecules on the researched phenomena would be excluded. The active site of AChE is quite stable and modification(s) in the ester and/or anionic sites should be evidenced by a depletion of enzyme activity [28,29]. Instead, access of organophosphates into a cavity with an active cleft would be a reason for the variation of AChE sensitivity to inhibition by an organophosphate [30,31]. However, this fact should be further clarified in the future. The effect of increased inhibitory potency of paraoxon after biosensor irradiation would be only estimated in the present study. A possible explanation could be found in a study by Weik et al. [35]. They found extensive sensitivity to X-rays of the His-440 residue in the active site of electric eel AChE. An impact of radiation would be also expected on the surface Glu-306, catalytically active Glu-327, and disulfide Cys-254-Cys-265 [35]. Dissociation of disulfides and modification of surface structural amino acids would be responsible just for increased penetration into the active site.

Cholinesterase based biosensors are an intensively researched group of biosensors. The current applications being widely published by many authors suggest promising performance and expected commercialization. Experiments presented here concerned the variation of cholinesterase activity when irradiated. The primary goal of the experiments was to assess the stability of immobilized AChE. Though something like a “biodosimeter” was expected, AChE was found to be stable, even under high doses of radiation. An unexpected effect was found when the pesticide paraoxon was assayed using the irradiated biosensors. The inhibitory efficacy of paraoxon was potentiated by irradiation. Though the achieved data are only preliminary, the positive effect on the assay is readily recognized.