Introduction

Recently, there has been a considerable interest in the prevention and detection of pesticide contamination which has proven to be a major environmental issue and has been linked to serious health problems including cancer, liver damage, kidney damage, reproductive difficulties, and nervous system effects. Among various classes of pesticides, organophosphate pesticide poisoning has been a major cause of concern the world over due to its severe effects on the nervous and reproductive systems of living beings. Phorate belongs to the class of organophosphorus pesticides (OPs) which have seen an augmented growth in its usage in the preceding years particularly in the agricultural fields. However, the increasing usage of phorate has posed a serious concern to the environment including humans and other mammals. The trace levels of OPs are known to significantly inactivate the catalytic activity of the enzyme acetylcholinesterase (AChE), an essential enzyme involved in the cholinergic functions, in the central and peripheral nervous systems. As a result of the inhibition of the enzymatic activity, acetylcholine starts accumulating in vivo thereby leading to serious complications like nausea, dizziness, confusion, and at very high exposures (such as accidents or major spills), respiratory paralysis and death.

Although several conventional techniques such as gas chromatography-mass spectrometry (GC-MS) and high-performance liquid chromatography (HPLC) exist for trace level determination offering high selectivity with a very low detection limit, these techniques are accompanied with numerous shortcomings. These include expensive instrumentation, complex sample pretreatment, longer analysis time, and requirement of trained personnel which confine their use for on-site screening of pesticide residues.

Recently, aptamers have emerged as promising tools due to their impending advantages over other recognition elements such as antibodies, enzymes, etc. Aptamers are single-stranded DNA or RNA sequences which bind to the target effectively with high affinity, selectivity, and sensitivity. They have gained much prominence as they can be generated in vitro by a technique named systematic evolution of ligands by exponential enrichment (SELEX) without compromising with the purity. Further, the use of aptamers is circumscribed not only to their applications in the areas of therapy, drug discovery, and target validation but they are also functional as recognition elements in sensors for various targets such as proteins, toxins, metal ions, DNA, and small molecules. However, the application of aptamers in pesticide detection is still limited which is supported by the scarcity of enough available reports in the literature.

Presently, colorimetric sensors have been reported to be dominating the residual analyte detection due to their benefits like rapidity, simplicity, cost-effectiveness, etc. In this context, various nanomaterials have appeared as promising means for colorimetric sensing but gold nanoparticles (AuNPs), in particular, have displayed best results owing to their simple preparation, high extinction coefficient, extra stability, easy functionalization, and distinct color changes. The red color of the well-dispersed AuNPs changes to blue upon aggregation, resulting in the shifting of the surface plasmon band to a longer wavelength, a feature that is employed widely in optical sensing. However, the challenge with the colorimetric sensors lies in their lack of selectivity. In view of the abovementioned points, we report a simple but effective and rapid method for the detection of phorate using aptamer-modified AuNPs. Basically, a pesticide-specific aptamer (apt) acted as the recognition element and the AuNPs modified with the aptamer was used to signal the presence of phorate (pesticide). The method is based on the principle that in the absence of phorate, the apt-AuNPs remain suspended even at high salt concentration due to the stabilization provided by the random coiled conformation of the aptamer. But, when phorate exists in the sample, it binds to the aptamer and forms a supramolecular complex which exposes the surface of the AuNPs to the electrolyte and results in the aggregation of the AuNPs. On the basis of the color change, quantification of phorate can be done efficiently. To the best of our knowledge, this is the first report where an aptamer is employed as a recognition element for the colorimetric detection of phorate in conjugation with the AuNPs.

Experimental

Materials and methods

Hydrogen tetrachloroaurate (III) trihydrate, trisodium citrate dihydrate, 2-mercaptoethanol, and an aptamer having the sequence 5′-AAG CTT GCT TTA TAG CCT GCA GCG ATT CTT GAT CGG AAA AGG CTG AGA GCT ACG C-3′ with disulfide (S–S) modification at the 5′ end were purchased from Sigma-Aldrich (India). All the reagents used were of analytical grade, and the experiments were performed in Milli-Q water and molecular-grade water having a resistivity of 18.2 MΩ cm. The glassware was rinsed in aqua regia prior to use.

Instrumentation

UV-vis spectra were recorded on a JASCO V-530 spectrophotometer. TEM measurements were carried out using a Hitachi H-7500 microscope. CD measurements were performed on a JASCO J-815 CD spectrometer.

Synthesis of gold nanoparticles

AuNPs were synthesized using reported procedure [1]. Briefly, 100 mL aqueous solution of HAuCl4·3H2O (0.25 mM) was heated to boil under constant stirring followed by the rapid addition of 2 mL trisodium citrate (34 mM). Initially, an intense dark violet color appeared and, finally, the solution turned red indicating the formation of AuNPs. Further, the solution was boiled for 10 min and then allowed to cool at room temperature (rt). The resulting gold nanoparticles were then characterized by a transmission electron microscope (TEM) and UV-vis spectrophotometer. The particle concentration was measured using a molar extinction coefficient of 2.4 × 108 M−1 cm−1 at 520 nm.

Modification of AuNPs with aptamer

The thiolated aptamer (2 μM, 1× TE buffer, pH 7.6) was mixed with 2-mercaptoethanol (1 %) and incubated for 1 h at rt in order to reduce the S–S linkage. After that, 100 μL of the phorate-binding aptamer (PBA) was added to the as-prepared 0.8 mL AuNPs (5 nM). The mixture was incubated at rt for 24 h. The excess of PBA was removed by centrifugation at 12000 rpm for 10 min, and the AuNPs were redispersed in molecular-grade water. The aptamer-modified AuNPs were then characterized by UV-vis spectroscopy and TEM.

Aggregation assay for phorate detection

Different concentrations of phorate were prepared in Milli-Q water. One hundred microliters of varying concentrations of phorate was added into 0.8 mL of apt-AuNPs, and the mixture was incubated for 1 h. Then, the solution was mixed with 100 μL NaCl (100 mM) and incubated for 5 min followed by the UV-vis measurements.

Analysis of phorate residues in spiked apple

To evaluate the feasibility of the proposed assay, an apple was selected as the matrix. The apple was cut and crushed into a homogenate. The apple juice was extracted with methanol and filtered to remove the solid part. The sample was then mixed with animal charcoal to eliminate colored impurities followed by filtration. Finally, the solvent was evaporated and the residue was diluted with water followed by spiking with phorate.

To analyze the phorate residues in the spiked samples, 100-μL spiked samples were incubated with 0.8 mL apt-AuNPs for 1 h at rt. Further, 100 μL NaCl (100 mM) was added and the samples were incubated for 5 min. Subsequently, the UV-vis spectra and color changes were recorded.

Results and discussion

Principle of the colorimetric assay

The detection of phorate using aptamer-conjugated AuNPs is based on the initial binding of the citrate-capped AuNPs to the thiolated aptamer via a strong Au–S bond [Scheme 1]. Since the covalent binding of Au–S is stronger than the electrostatic attraction of Au–citrate, the aptamer replaces the citrate ligands thereby stabilizing the AuNPs. In the absence of phorate, the aptamer acquires a random conformation as a result of which the negatively charged bases of the aptamer project outwards. These bases prevent the aggregation of the particles via electrostatic repulsions and, thus, provide stability to the AuNPs. Consequently, the addition of electrolyte is unable to induce the aggregation of AuNPs, thereby keeping them well dispersed and red in color. However, when phorate is added into the solution, the aptamer specifically binds to phorate, leading to the change in the conformation of the aptamer from a random coil to a rigid one. As a result, the rigidity of the supramolecular complex formed between phorate and aptamer inhibits the nucleic bases from projecting and, therefore, exposes the AuNPs to the electrolyte, leading to the aggregation of the nanoparticles imparting a blue color to the solution.

Scheme 1
scheme 1

Schematic illustration of the principle of the colorimetric detection of phorate utilizing aptamer-modified gold nanoparticles. The red color of the well-dispersed apt-AuNPs changes to blue in the presence of phorate upon electrolyte addition

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Initially, the aggregation of AuNPs was investigated under different conditions. The citrate-stabilized AuNPs were originally red in color in their dispersed state; however, the addition of NaCl screened the electrostatic repulsions due to the presence of opposite charges and aggregated the AuNPs, giving them blue color. The UV-vis spectrum of AuNPs in the presence of NaCl showed the decrease in the characteristic absorption of AuNPs at 520 nm and led to the emergence of an absorption band around 700 nm. However, in the presence of PBA, the stability and color of the AuNPs were maintained which is exhibited by the sharp peak at 520 nm. This suggests that the aptamer serves as a stabilizer and protects the AuNPs from salt-induced aggregation. On the contrary, in the presence of phorate, the strong affinity of phorate and PBA resulted in decrease in the intensity of the peak at 520 nm and appearance of a new band around 620 nm. This was accompanied by the change in color from red to blue.

The aggregation phenomenon of the AuNPs was further confirmed by TEM images. The well-dispersed nature of the aptamer-modified AuNPs is evident from Fig. 1A where the particles are 10 nm in diameter and are spherical. However, the presence of phorate led to the change in the conformations of the aptamer due to the interaction of phorate and PBA. Therefore, the addition of NaCl screened the repulsions between the particles and brought the aptamer-modified AuNPs closer, as a result of which the color of the nanoparticles turned blue due to aggregation (Fig. 1B).

Fig. 1
figure 1

TEM image of aptamer-modified AuNPs (A) and apt-AuNPs in the presence of 2 μM phorate and 100 mM NaCl (B)

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Optimization of reaction conditions

In order to develop a highly effective aptasensor for the detection of phorate, all the reaction conditions of the assay were carefully optimized. Firstly, the concentration of PBA for conjugation with AuNPs was varied from 0.1 to 2 μM. It was observed experimentally that the AuNPs conjugated to lower PBA concentrations, i.e., 0.1, 0.5, and 1.0 μM, turned slightly pink in color on the addition of NaCl whereas the AuNPs with 2 μM PBA remained red. Therefore, the higher PBA concentration was selected for further experiments. Then, the concentration of NaCl was optimized since it has a pronounced effect on the stability of particles. Experiments were carried out with varying concentrations of NaCl such as 50, 100, 200, 300, and 500 mM. The results obtained showed that the aptamer-modified AuNPs were stable up to 100 mM NaCl concentration while the higher NaCl concentrations led to the aggregation of the particles. Hence, 100 mM NaCl was chosen for further analysis.

Detection of phorate

After careful optimization of the reaction conditions, the sensitivity of the proposed aptasensor was thoroughly investigated. Different phorate concentrations ranging from 0.01 nM to 10 μM were treated with the apt-AuNPs and NaCl, and the resulting absorbance spectra were recorded. The UV-vis spectra of the AuNPs showed that with the increase in phorate concentration, the characteristic absorbance at 520 nm decreased whereas the absorbance band around 620 nm increased progressively which is due to the aggregation of the nanoparticles (Fig. 2a). The results proved that the specific binding of the PBA increased with the increase in phorate and, therefore, resulted in the aggregation of the AuNPs. The ratio of A620/A520 was found to be linear in the concentration range of 0.01 nM to 1.3 μM and became constant afterwards because of the precipitation of the aggregated AuNPs (Fig. 2b). Accordingly, the color of the solutions changed from red to purple to blue.

Fig. 2
figure 2

UV-vis spectra of apt-AuNPs in the presence of different phorate concentrations and 100 mM NaCl (a), ratio of absorbance of apt-AuNPs at 620 and 520 nm versus concentration of phorate (0.01, 100, 300, 500, 700, and 900 nM; 1.1, 1.3, 2, 4, 6, 8, and 10 μM) (b), and UV-vis spectra of apt-AuNPs in the presence of different pesticides (5 μM). Each point is the average of three measurements and error bars indicate standard deviation. Insets show their corresponding pictures

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Selectivity of the aptasensor for phorate

Due to the presence of different chemical entities in the environment, the selectivity of any sensor is very crucial. To evaluate the selectivity of the proposed sensor, cross-reactivity studies were carried out using other interfering pesticides such as atrazine, chlorsulfuron, 2,4 D, diuron, and ethyl parathion. The experiments demonstrated that only phorate could induce the aggregation of AuNPs and change the color of AuNPs from red to purple since the aptamer is specific for phorate only. All the other interfering pesticides were unable to aggregate the AuNPs, and the solution remained red in color. Figure 2c clearly shows that aggregation occurred only in the case of phorate and the response of the sensor towards other pesticides was significantly low. These results clearly ascertain the high selectivity of the present aptasensor.

The limit of detection (LOD) of the proposed assay was calculated to be 0.01 nM using the formula 3α/slope, where α is the standard deviation of the instrument. The LOD obtained was significantly lower than the methods already available for phorate detection, establishing the high detectability of the proposed assay (Table 1). Although the linearship of 0.9322 as obtained from the measurements is not high, the ease of experimental procedure, low limit of detection, low cost, rapidity, selectivity, and absence of highly sophisticated instrumentation renders this assay a highly selective and economical method of detection of organophosphorus pesticide phorate.

Table 1 Comparison of the proposed assay with other techniques for the detection of phorate
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Analysis of phorate in real samples

In order to check the applicability of the aptasensor in real samples, the proposed assay was applied to the apple matrix. The apple juice was spiked with different concentrations of phorate, and the analysis was performed. The concentration of phorate obtained from the regression curve was found to be closer to the added value (Table 2). This further confirmed the reliability of the assay and suggested that the method can be applied to real sample as well. The results obtained were further compared with conventional techniques such as HPLC using Waters HPLC system having the 2996 photodiode array detector. A C-18 column (250 × 4.60 mm, 5 μm, Alltima) was used for the measurements with water/acetonitrile as the mobile phase. It was found from the experiments that the concentration which was non-detectable in HPLC could be easily detected with the given method.

Table 2 Determination of phorate residue in spiked apple
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Conclusion

In summary, a colorimetric sensor based on an aptamer and AuNPs was successfully developed for the rapid and specific detection of phorate, an extremely toxic organophosphate pesticide. The detection exploits the specific binding of the aptamer and phorate and the color changes associated with this interaction. In the absence of phorate, the aptamer-modified AuNPs remain dispersed and red in color even in the presence of a high electrolyte concentration whereas on the introduction of phorate, the state of the nanoparticles changes to the aggregated one and the solution appears blue in color. The method exhibited high selectivity as it was able to selectively detect phorate within a mixture of pesticides with a LOD of 0.01 nM. Moreover, the method was also applicable to a real sample signifying the applicability of the assay. The proposed colorimetric sensor is devoid of the use of any solvent, complex sample preparation, enzyme, light-sensitive reagents, or sophisticated instruments, thus offering its potential for the rapid and efficient on-field monitoring of phorate residues in particular and organophosphates in general.