Abstract
This paper presents the results on the fabrication of highly sensitive fluorescence biosensors for pesticide detection. The biosensors are actually constructed from the complex of quantum dots (QDs), acetylcholinesterase (AChE) and acetylthiocholine (ATCh). The biosensor activity is based on the change of luminescence from CdSe and CdTe QDs with pH, while the pH is changed with the hydrolysis rate of ATCh catalyzed by the enzyme AChE, whose activity is specifically inhibited by pesticides. Two kinds of QDs were used to fabricate our biosensors: (i) CdSe QDs synthesized in high-boiling non-polar organic solvent and then functionalized by shelling with two monolayers (2-ML) of ZnSe and eight monolayers (8-ML) of ZnS and finally capped with 3-mercaptopropionic acid (MPA) to become water soluble; and (ii) CdTe QDs synthesized in aqueous phase then shelled with CdS. For normal checks the fabricated biosensors could detect parathion methyl (PM) pesticide at very low contents of ppm with the threshold as low as 0.05 ppm. The dynamic range from 0.05 ppm to 1 ppm for the pesticide detection could be expandable by increasing the AChE amount in the biosensor.
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1. Introduction
Pesticides such as organophosphorus (OP) compounds, carbamates or neonicotinoids are widely used in agricultural production due to their high efficiency for insect killing, easy synthesis and low cost. It is well known that such pesticides are effectively inhibiting the activity of AChE, the most important enzyme involved in nerve transmission. The principal role of AChE is the termination of nerve impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine (ACh), each molecule of AChE degrades about 25 000 molecules of ACh per second. Therefore, the inhibition of AChE by pesticide leads to in vivo accumulation of ACh, and then results in serious impairment of nerve functions or even death. Over time, pesticides used reside not only on agricultural products such as fruit, rice and tea but have also accumulated in cultivation soil. Residues, even at very low concentrations of highly toxic OP-based pesticides, therefore cause serious problems to human health and severely threaten public safety [1–13].
In order to control the residual pesticides in agricultural products to avoid possible harm to humans and the environment for life, it is obvious that techniques for detecting low concentrations of OPs must be developed. Currently, there have been various techniques to detect pesticides, such as electrochemistry, colorimetric probes, liquid or gas chromatography [4,11,12]. However, all these demand expensive instruments and well-trained personnel, and are time-consuming in operation. That is the reason why people are still looking for other low-cost, fast and easy techniques to detect the residual pesticides for applications in agricultural production and environmental protection. Recently, some reasonable techniques have been approached based on the exploitation of the optical properties of semiconductor quantum dots (QDs) [14–17]. One kind of fluorescence biosensor has been realized by the exploitation of the luminescence change depending on the pH of the medium surrounding the QDs [14–16] that indicates the pesticide content; another was based on the resonance energy transfer from QDs to molecular complexes dithizone (DZ) that gives rise to a luminescence on–off biosensor [17]. These are hoped to be realized as sensitive, fast, portable and easy-to-use techniques. However, there are still many questions related to the reliability of such biosensors in practical applications.
In this paper, we present the fabrication of biosensors following the principle of specific inhibition of the enzyme AChE by pesticides that consequently causes a change in the hydrolytic rate of ATCh followed by a change in the photoluminescence (PL) intensity of CdSe or CdTe QDs. The core/shell/shell (CSS) structure of CdSe/2-ML ZnSe/8-ML ZnS QDs and the core/shell (CS) one of CdTe/CdS QDs were used to fabricate our biosensors. Influences of the ratios of QDs to other constituents such as ATCh and AChE on the performance of biosensors were systematically studied to determine good technical parameters for the biosensors. The sensitivity and dynamic range of our biosensors for the detection of residual pesticides were checked with parathion methyl (PM) pesticide showing that PM at very low content of 0.05 ppm could be detected.
2. Experimental
2.1. Materials and methods
In order to fabricate the fluorescence biosensors, we have used three constituents that are the enzymes AChE, ATCh and highly luminescent QDs (CdSe/ZnSe/ZnS CSS and CdTe/CdS CS QDs). The enzymes AChE (EC 3.1.1.7, specific activity 500 Units/0.7mg) and S-acetylthiocholine iodide (ATChI) were purchased from Sigma-Aldrich. ATCh was prepared from ATChI according to the method in [8]. CdSe/ZnSe/ZnS CSS QDs were synthesized in high-boiling organic solvent by using the heating-up method [6,7]. This CSS structure was prepared first with the core CdSe QDs, then 2-ML ZnSe shell and then coating with 8-ML ZnS shell. Because the QDs were synthesized in non-polar high-boiling organic solvents they are not water-soluble. Therefore, in order to be able to use these QDs in fabrication of biosensors for pesticide detection, a surface modification by ligand exchange with MPA must be done to make these QDs water-soluble. The size of CdSe QDs was controlled by the growth/heating time, typically in minutes to hours. We used CdSe QDs that have the size of 4.5 nm (synthesized at 210 °C for 18 min in trioctylphosphine oxide and hexadecylamine) to emit ∼ 610 nm (the red spectral region) though the biosensor does not require QDs emitting at specific wavelengths. Along with our work, we see that CdTe QDs synthesized directly in aqueous phase could be used more easily and efficiently to fabricate biosensors. Details of the syntheses of high-quality CdTe/CdS CS QDs to strongly emit PL in the green-red spectral range have been described elsewhere [6]. For the purpose of comparison, we used CdTe QDs that have the size of 3.8 nm (synthesized at 120 °C for 30 min in distilled water, with the Cd:Te:MPA ratio of 1:4:1) to emit around the same wavelength, ∼ 610 nm.
For testing the biosensor activity, pesticide PM which is an organophosphate compound was chosen. PM was purchased from Fluka.
The absorption and PL spectra were taken by using a Varian Cary 5000 UV-Vis-NIR spectrophotometer and an iHR550 (Horiba) spectrometer equipped with a thermoelectrically cooled Si-CCD camera (Synapse), respectively. In the PL measurement, a 405 nm diode laser was used as the excitation source.
2.2. Fabrication of the fluorescence biosensor for pesticide detection
In this paper, we just present a kind of fluorescence biosensors in the liquid cell-form. The principle of the actual biosensor is the measure of the PL intensity of QDs as a function of pH that correspondingly reflects the pesticide content. Normal 1 × 1 × 4.5 cm3 organic glassy cuvettes were used to contain the solutions prepared by mixing well the enzymes AChE, ATCh and CdSe or CdTe QDs. In the mixed solution, AChE catalyzes the hydrolysis of ATCh to produce TCh, which bears an additional thiol group (–SH) to make the possible increase of the pH surrounding the CdSe or CdTe QDs. It is quite possible that AChE catalyses the hydrolysis of ATCh producing TCh and acetic acid or acetate in a reversible way, meaning that if AChE stops its catalytic activity, ATCh could be reproduced from TCh and acetic acid or acetate. In this biosensor structure, ATCh acts as an indicator for the presence of the AChE enzymatic activity, while the CdSe or CdTe QDs act as the luminescence indicators for the hydrolysis of ATCh. Eventually, the luminescence from CdSe or CdTe QDs can indicate the AChE enzymatic activity or correspondingly the pesticide content, because when adding pesticide into the mixed solution (biosensor, composed of the enzymes AChE, ATCh and CdSe or CdTe QDs), the pesticide would bind to AChE to inhibit the AChE enzymatic activity. The amounts and ratios of each constituent above were designed depending on the sensitivity and dynamic range of the pesticide detection.
In normal biosensor fabrication, we used directly the CdSe/ZnSe/ZnS CSS QDs or CdTe/CdS CS QDs. These QDs were cleaned just by centrifugation (5800 rpm for 10 min) to get the clear colloidal QDs solutions and then dispersed in phosphate buffered saline (PBS) solution (pH = 7.4). The amount of 16.4 units ml−1 AChE was dissolved in 1 ml of the QDs solution (typically absorbance of 10−4 corresponding to about 1012 QDs per ml) containing 25 μl ACTh (0.12 mM). The mixed solution was incubated at 37 °C for 30 min with the assistance of an ultrasonic machine (45 kHz) to mix well all the three constituents of the biosensor. At this stage, we have finished the fabrication of biosensor which was ready to detect pesticide. PMs in distilled water of various concentrations, from 0.05 ng/ml (0.05 ppm) to 3 ng/ml (3 ppm), were prepared to check the function of the fabricated biosensors. The PL measurements were carried out 5 min after adding pesticide into biosensor at room temperature.
3. Results and discussion
Figure 1 shows the absorption and PL spectra of the CdSe/ZnSe/ZnS CSS QDs and CdTe/CdS CS QDs which were used to fabricate the fluorescence biosensors. One can see that the excitonic absorption of CdSe/ZnSe/ZnS CSS QDs becomes less pronounced after doing the ligand exchange to make these QDs water soluble. However, the PL is still very strong. For the CdTe/CdS CS QDs the excitonic absorption is clear showing the good quality of the aqueous synthesized QDs.
Figure 1. Absorption (solid) and PL (dotted) spectra of CdSe/ZnSe/ZnS (upper) and CdTe/CdS (lower) QDs.
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Standard imageWe have fabricated two kinds of the QDs-AChE-ATCh-based biosensors using two kinds of QDs: (i) CdSe/ZnSe/ZnS CSS QDs and (ii) CdTe/CdS CS QDs. This is because, if both QDs provide the same function in the fluorescence biosensors, we should use the CdTe/CdS QDs instead of CdSe/ZnSe/ZnS CSS QDs for greater convenience. In order to have a good set of the technical parameters for fabricating the biosensor, we first study the influence of ATCh or AChE or PM on the PL of QDs. The results show that almost no change was observed in the PL spectra of both CdSe/ZnSe/ZnS CSS QDs and CdTe/CdS CS QD. Next, the mixed solution of QDs and ATCh or AChE added with PM showed no change in the PL spectra. These are quite understandable because the pH of the media surrounding the QDs was not changed with PM or separate ATCh or AChE. The last experiment shows the great decrease of the PL intensity from QDs after adding both ATCh and AChE. This is because AChE catalyzed the hydrolysis of ATCh to produce ACh with more SH– group to increase the local pH. The quenching and blue shift of the PL from the QDs-ATCh mixture upon increasing the AChE content was observed to be similar to the results presented in [8]. One can figure out that now if PM is added to inhibit the AChE enzymatic activity, the PL intensity from QDs can be recovered. Figure 2 shows such a nice picture for the fluorescence biosensor fabricated from the CdSe/ZnSe/ZnS CSS QDs. By adding both AChE (1 unit) and ATCh (in the QDs:AChE:ATCh ratio of 1:1.5:1) the PL intensity decreased greatly; then by adding PM to inhibit the AChE enzymatic activity consequently to eliminate the hydrolysis of ACTh that makes a respective increase in the PL intensity. The sensitivity of 0.05 ppm PM has been achieved that corresponds to the limit detection of the EU standard 2010 (the EU Reg. No 600/2010 of 8 July, 2010) and much lower than the residual amount around ppm that is allowed to be residual in foods. The detection dynamic range of this biosensor is between 0.05 ppm and 0.5 ppm. This range is relative to the increase of the QDs' PL intensity with adding PM until saturation of PL. The saturation of the PL intensity corresponds to the inhibition of all enzymes AChE that exist in the biosensor that no more hydrolysis of ATCh takes place to make any change in the local pH of medium surrounding the QDs. The dynamic range for pesticide detection can be expandable by increasing the AChE content as presented below.
Figure 2. PL spectra (under the 405 nm laser light excitation) of CdSe-AChE-ATCh as a biosensor to detect different PM contents. The inset shows the relationship between the PL intensity and the PM contents.
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Standard imageWe now study the CdTe/CdS QDs-based biosensor. The effect of ATCh, AChE or PM on the PL of CdTe/CdS QDs was separately examined showing no change of the PL spectrum from CdTe/CdS QDs similar to the case of CdSe/ZnSe/ZnS CSS QDs. This means that there is no chemical interaction between QDs and ATCh, AChE or PM separately. However, with adding both ATCh and AChE into CdTe/CdS QDs the PL intensity of these QDs significantly decreased. Using 1 unit of AChE in the CdTe/CdS QDs-ATCh-AChE-based biosensor, we observed an increase of the PL intensity of CdTe/CdS QDs with increasing PM content, similar to the biosensor using CdSe/ZnSe/ZnS CSS QDs. The dynamic range for the detection of PM of the biosensor using 1 unit AChE is in between 0.05 ppm and 0.5 ppm. As mentioned above, this dynamic range can be expandable by increasing the AChE content (also meaning the increase of the AChE:QDs ratio). Figure 3 shows the similar behaviors of the biosensor made from CdTe/CdS QDs compared to the one made from CdSe/ZnSe/ZnS CSS QDs with adding 3 units of AChE to expand the dynamic range for the PM detection to be between 0.05 ppm and 1 ppm. Figure 4 shows the results from the two biosensors fabricated with the two different AChE amounts of 1 and 3 units, respectively.
Figure 3. PL spectra (under the 405 nm laser light excitation) of CdTe-AChE-ATCh as a biosensor to detect different PM contents. The inset shows the relationship between the PL intensity and the PM contents.
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Figure 4. PL intensity of CdTe-AChE-ATCh (under the 405 nm laser light excitation) with the two different AChE amounts of 1 and 3 units as functions of the PM contents.
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Standard imageIt seems that CdTe/CdS QDs synthesized in aqueous phase are very appropriate for the biolabeling application. Particularly, the aqueous synthesized CdTe/CdS QDs are very suitable for fabrication of a biosensor to detect residual pesticides. In fact, AChE catalyses the hydrolysis of ATCh, producing TCh and acetic acid or acetate in a reversible way, meaning that if AChE stops its function, ATCh could be reproduced from TCh and acetic acid or acetate. In case all the enzymes AChE of a biosensor have been inhibited, the addition of a new amount of AChE can make such a biosensor reactive again. This phenomenon was recently observed and is now under study.
4. Conclusion
In conclusion, we have successfully fabricated fluorescence biosensors using CdSe/2-ML ZnSe/8-ML ZnS CSS QDs or CdTe/CdS CS QDs in combination with ATCh and AChE. The biosensor activity is based on the change of luminescence from CdSe and CdTe QDs with pH, while the pH is changed with the hydrolysis rate of ATCh catalyzed by the enzymes AChE, whose activity is specifically inhibited by pesticides. These highly sensitive fluorescence biosensors showed good performance in detection of the PM pesticide at very low contents of ppm with the threshold as low as 0.05 ppm. The dynamic range for the PM pesticide detection between 0.05 and 1 ppm has been achieved and it could be expanded by increasing the AChE amount in the biosensor.
Acknowledgments
The authors sincerely thank Acad. Nguyen Van Hieu for his helpful discussions and his generous support. This work was financially supported by MOST Vietnam (contract 1/2/742/2009/HD-DTDL). The authors also thank the National Key Laboratory for Electronic Materials and Devices-IMS for the use of facilities.