Study on Fluorescence Spectrum of Pesticide and Serum in Interaction

The fluorescence spectrum of different serum samples added at the same concentrations with different pesticides (seed coated agent, herbicide and the flapping lice spirit) has been studied when 260, 280 and 320 nm, respectively exciting lights are used to excite the serum. It is found by experiment that when the same pesticide is added to the serum in different biochemical indexes, the fluorescence spectrum peak changes differently. The red shift occurs for the position of the fluorescence peak when the different pesticides are added to the serum in the same biochemical index. For the serum in different biochemical indexes added with the pesticides, the fluorescence intensity changes differently. The aim of study was to determine the type of pesticides based on the change of the serum fluorescence intensity. The experimental results show that pesticides have a great influence on the fluorescence of the serum in different biochemical indexes. The further research results will provide an experimental basis for first aid and drug use for patients who are suffered from pesticide or herbicide poisoning in different biochemical indexes.


INTRODUCTION
The fluorescence spectrometry as a kind of analysis method is widely used in various fields of industry, agriculture, medicine, health, judicial authentication and scientific research.Detecting pesticide residues pollution in combination with optical fiber sensing and computer signal processing technology is becoming a research focus in the world in this field (Xie et al., 2010;Wang et al., 2010;Ma et al., 2006).The research on fluorescence analysis of pesticides was started by foreign scholars in the 1990s.They had successfully detected fluorescence spectrum of several pesticides by making use of natural fluorescence properties of some pesticides with the help of some fluorescent dyes.Meanwhile, they developed various analyzers based on optical fiber sensing technology (Tanojo et al., 2000;Armenta et al., 2007).Harris et al. (1998) determined the pesticide residues of dichloro ben oxyacetic acid in water using the optical fiber fluorescence immunoassay method.Hassoon and Schechter (2000) developed a fluorescence probe for measurement of DDT pesticides and determination of DDT residues in soil.JiJi et al. (1999) applied the Excitation Emission Matrix (EEM) fluorescence spectrum for the quantitative detection of trace PAHs and carbamate pesticide residues.At present, the rapid detection technology of pesticide residues based on laser-induced fluorescence spectrum analysis is just in a stage of principle and experimental research at home and abroad.Sun et al. (2003) used laser-induced fluorescence technology to measure the fluorescence spectrum of five pesticides such as tetrachlorvinphos and conducted a preliminary qualitative research on pesticide fluorescence detection.Che and Wang (2004) has developed a fiber optic spectrometer to monitor imidacloprid pesticide.Wang et al. (2006) detected the carbamate pesticide residues using the three-dimensional fluorescence technology.Zhang et al. (2006) studied the carbaryl spectral characteristics by the synchronous fluorescence spectrum analysis and determined its concentration.Most of these pesticides in question are organic compounds.As molecular structure of different pesticides is different, each fluorescent material can give off specific fluorescence spectrum.In the same excitation, different pesticides give off the fluorescence spectrum in different intensity and show different shapes.Because the fluorescence spectrum of different pesticides is significantly different in distribution, time delay and formation efficiency, the kind and concentration of pesticides can be detected by studying photo-induced fluorescence characteristics (Yang and Gong, 2006;Li et al., 2004).This research provides a good foundation for fluorescence spectrum analysis applied for real-time and rapid detection of pesticide residues as well as further development of fluorescence spectrum technology.Reports of pesticide and serum interaction are rare though many spectrum detections related to pesticides have been reported.This study deals with the spectrum law of interaction between pesticides and serums in different biochemical indexes.

SAMPLES AND METHOD
 Experimental serum samples were taken from healthy male patients suffered from high blood lipid and high blood glucose a total of 54 subjects by the vein blood sampling when they were in an empty stomach in early morning.Take the upper serum and prepare the 1:20 serum solution with pure water for testing use. The pesticides for the experiment were: Imidacloprid: 10% wettable powder (made by Jiangsu Changqing Agrochemical Co., Ltd.); dichloro•Benzyl: 36% wettable powder (Jiangsu Changqing Agrochemical Co., Ltd.); and 35% seed coating agent (EN Industry Corporation).In the experiment, dissolve 0.2 g active compound by weighing pesticides with distilled water and dilute the solution to 100 mL.After that, take the supernatant liquid and put it into a water bath at a constant temperature of 25°C for use. In the experiment, the Shimadzu RF5301 fluorescence spectrophotometer was used to detect the fluorescence spectrum of selected serum samples and serum samples containing pesticides.Take a 3 mL tested sample with a cuvette in the test.According to the early stage of the experiment, studied the effect of wavelength on serum fluorescence excitation (Published in 2008, the tenth period《spectroscopy and spectral analysis》).Therefore, this experimental selected excitation wavelengths respectively were 260, 280 and 320 nm, respectively the accepted wavelength ranges from 280 to 600 nm and the slit width of excitation and emission was 3 nm.The sampling interval was 0.5 nm, with a medium speed automatic scanning every 5 nm.

RESULTS AND DISCUSSION
In order to study effect of pesticides in different biochemical indexes of serum, the fluorescence spectrum of samples in normal biochemical indexes, high blood glucose, high cholesterol and high triglycerides was measured separately by spectrometer.Serum samples respectively were added to pesticides at the same concentration and the fluorescence spectrum was measured.The experimental results are shown in Fig. 1 to 3. According to the variation of the fluorescence intensity of the spectral lines, the fluorescence intensity changes of the different serum samples added with different pesticides at the same concentration was listed after the spectral analysis as shown in Table 1 to 3   (b) The fluorescence intensity changes of the serum samples added with pesticides at the same concentration (Ex: 260 nm)       The red shift occurs for fluorescence peak and the amount of motion for the serum in different biochemical indexes is different after the serum is added with pesticides as shown in Table 1.
Especially 2 nm blue shift occurs for high triglyceride serum after seed coating agent is added.After added with herbicides, the high glucose serum has a smaller peak at 327 nm than that of normal serum, while the high blood lipid (including high cholesterol and triglyceride) serum has a bigger peak than that of normal serum.After different pesticides are added, the fluorescence intensity is different as shown in Fig. 2a. Figure 3a shows, the fluorescence intensity increases at near 330 nm after the seed coating agent is added in serum.This is because when the seed coating agent is excited with 260 nm excitation light, the fluorescence also occurs at near 330 nm, resulting in an increase of fluorescence intensity rather than quenching at wavelength.After seed coating agent is added in serum in different biochemical indexes, there is a different increase of fluorescence intensity.Compared with the normal serum, the serum of patients with hyperglycemia and high cholesterol has a smaller increasing intensity, indicating that added seed coating agent has a great impact.After herbicides and the flapping lice spirit are added, the fluorescence intensity is weakened  The fluorescence intensity changes greatest for high blood glucose and it is followed by the high cholesterol; the fluorescence intensity changes smallest for high triglycerides.This further indicates the serum in different biochemical indexes is impacted by herbicides differently.Compared with 260 nm excitation light, the fluorescence intensity increases for the normal and high blood glucose and high cholesterol serums after the serum in different biochemical indexes is added with the flapping lice spirit, but the fluorescence intensity decreases a little for high triglyceride serum.This suggests that the fluorescence intensity of the flapping lice spirit exerts a strong impact on serum. It is seen from Fig. 3 that the fluorescence spectrum changes differently when 320 nm light excitation serum and different pesticides are added into the same serum and the fluorescence intensity at 360 nm increases.The blue shift occurs for the fluorescent peak and the quantity of motion is different after the serum in different biochemical indexes is added to pesticides as shown in Table 3.The fluorescence spectrum changes after the same pesticide is added to the serum in different biochemical indexes.After normal serum is added in the flapping lice spirit, there is a peak at 448 nm, but no peak for high blood glucose and high blood lipid serums, instead, forming a wide shoulder peak of 404-440 nm.After the high cholesterol is added in seed coating agent, the shape of its spectral lines changes significantly compared with that of normal serum, forming a wide shoulder peak of 400-436 nm.There is an intersection point between the spectra of the serum added and seed coating agent, but the position of the intersection is different for fluorescence spectrum of the serum in different biochemical indexes.It is intersected at 450 nm for the fluorescence spectrum of normal serum, 450 nm for high blood glucose serum, 470 nm for, high cholesterol serum and 427 nm for high triglyceride serum.The fluorescence spectrum of the serum added with herbicide is intersected with that of normal serum at 404 nm, 421 nm for high blood glucose serum, 398 nm for high cholesterol serum and 402 nm for high triglycerides.
It is known from Fig. 3c that the serum fluorescence intensity increases at 362 nm when 320 nm light excitation serum is added with different pesticides.The degree of enhancement is different for different pesticides.The fluorescence intensity increases greatest after the serum is added with the flapping lice spirit.This is mainly because when the 320 nm light is excited, the pesticide has the strongest fluorescence itself and the degree of fluorescence intensity quenching is far less than the glow of pesticides after the serum is added with pesticides.The fluorescence intensity increases to some extent when the serum is added with seed coating agent and the intensity is minimum for the herbicide added.The fluorescence intensity increases greatest for high blood glucose serum added with the flapping lice spirit; it is followed by the high triglycerides serum and the last one is high serum cholesterol serum.It suggests that the biochemical indexes of the serum added with pesticide still exert a great impact on the fluorescence of serum. It is seen from Fig. 4 that the type of pesticides added in the serum can be judged based on changes of the serum fluorescence strength.When the 260 nm excitation light is used to excite the serum, the seed coating agent has the strongest fluorescence intensity after the pesticide is added.The herbicide has the strongest fluorescence quenching to the greatest extent, but the flapping lice spirit has a little fluorescence quenching.Especially when the herbicide is added, the high blood glucose serum has the less fluorescence strength quenching than normal serum and the high blood lipid serum has the greater fluorescence strength quenching.When the serum is excited with the 280 nm light, the fluorescence strength increases greatest for seed coating agent when the pesticide is added, the fluorescence strength increases less for the flapping lice spirit, but the strength increases weakly for herbicide.When the serum is excited with the 320 nm, the fluorescence strength increases greatest for seed coating agent when the pesticide is added, the fluorescence strength increases less for the flapping lice spirit, followed by seed coating agent and least strength for herbicide.
 When join pesticides into the serum, the molecule of serum and pesticides will interact each other and bring fluorescence quenching, so the strength of serum fluorescence will be lower.Since the different serum (normal, high blood glucose, high cholesterol and high blood lipid) has different molecular concentration, so the same pesticides interact with different serum, the degree of fluorescence quenching will be different; the law of the fluorescence spectrum is also different.The fluorescence will be stronger after put pesticides into the serum, perhaps because seed coating agent, herbicide and the flapping lice spirit these three kinds of pesticides all have benzene ring and conjugated double bond, in some condition will be excited and bring fluorescence, lead by the superposition of two fluorescence.That is to say after pesticides join into serum the degree of the fluorescence strength quenching will be smaller than pesticides luminescence.

CONCLUSION
It is known from the experimental results that seed coating agent, herbicide and the flapping lice spirit which contain carbonates pesticide have strongly fluorescent characteristics, which have a greater influence on blood fluorescence.In this study, the 260, 280 and 320 nm excitation lights are used to excite the serum and the fluorescence spectrum is studied for different serum samples added with different pesticides at the same concentration.The fluorescence spectrum changes differently when the same pesticide is added to the serum in different biochemical indexes.At the same time, the fluorescence spectrum is also different when different pesticides are added to the same serum sample.The red shift occurs for the position of the fluorescence peak.The variation of serum in different biochemical indexes is different after the serum is added to pesticides.According to changes of the serum fluorescence strength, the type of pesticides added to the serum can be judged.This experiment indicates that the biochemical indexes of the serum have a great influence on its fluorescence after the serum is added in the pesticides.However its mechanism of action should be further studied.Its deeper research will provide experiment basis for the first-aid and drug use for patients who are suffered pesticide poisoning in different biochemical indexes.

Fig. 1 :
Fig. 1: The fluorescence spectrum of the serum samples added with pesticides at the same concentration (Ex: 260 nm) . The experimental results are analyzed as follows: Figure1shows that the fluorescence spectra change differently when 260 nm light excitation serum and the same pesticides are added to the serum in different biochemical indexes.When the different pesticides are added to the same serum, the fluorescence spectrum changes also differently.
Fig. 2: The fluorescence spectrum of the serum samples added with pesticides at the same concentration (Ex: 280 nm) Table 2: (a) The fluorescence intensity changes of the serum samples added with pesticides at the same concentration (Ex: 280 nm)

Fig. 3 :
Fig. 3: The fluorescence spectrum of the serum samples added with pesticides at the same concentration (Ex: 320 nm) Table 3: (a) The fluorescence intensity changes of the serum samples added with pesticides at the same concentration (Ex: 320 nm)

Fig. 4 :
Fig. 4: The fluorescence intensity changes of different serum samples added with different pesticides at near 330 nm in different degrees.Obviously, herbicides have a big impact and the flapping lice spirit has a small impact on serum.For serum in different biochemical indexes after added herbicides, the fluorescence intensity decreases in different degrees.Compared with normal serum, hyperglycemia is weakened in a smaller degree than that of normal one and high cholesterol serum decreases in a larger degree than normal one, indicating the serum in different biochemical indexes is impacted by herbicides to different extents.It may be because pesticide molecules are reacted with serum protein molecules after they come into the serum, so that the fluorescence quenching occurs.Compared with normal serum, patients' serum molecular structure and environment change a little, resulting in fluorescence quenching to some extent when

Table 1 :
(a)The fluorescence intensity changes of the serum samples added with pesticides at the same concentration (Ex: 260 nm)