†These authors contributed equally.
Academic Editor: Soo-Jin Choi
Background: Diazinon is a widely used organophosphorus neurotoxic insecticide. It is a common environmental contaminant and a hazardous agri-waste. Its detection is critical to control entry into food systems and protect the environment. Methods: In this study, three single-stranded DNA aptamers specific for diazinon were discovered using the systematic evolution of ligands by the exponential enrichment (SELEX) process. Since aptamer-based sensors are quick and straightforward to analyze, they could potentially replace the time-consuming and labor-intensive traditional methods used for diazinon detection. Results: Here, we show the engineering of novel sensors for diazinon detection with a high affinity (Kd), specificity, and high sensitivity at the ppb level. Moreover, the aptamers were helpful in the simultaneous detection of two other structurally relevant insecticides, fenthion, and fenitrothion. Furthermore, the real vegetable and fruit samples confirmed the specific detection of diazinon using DIAZ-02. Conclusions: We developed novel biosensors and optimized the assay conditions for the detection of diazinon from food samples, such as vegetables and fruit. The biosensor could be adopted to analyze toxicants and contaminants in food, water, and nature as point-of-care technology.
Diazinon is a broad-spectrum organophosphorus insecticide and a neurotoxic compound. Since its registration (1956) for commercial use in the US, it has become a top-ranked insecticide in consumption [1]. The diazinon and its products are available under various formulations and trade names, including Alfatox, Basudin, Cekuzinon, Diazol, Gardentox, and Knoxout [1], used to kill several classes of insects. In agriculture, it has been helpful to control the pests of the soil, field crops, vegetables, and livestock. In residential areas, it is used to protect houses, gardens, and pets from aggressive insects and insect-borne diseases. The mode of action of diazinon targets the central nervous system via the blocking of acetylcholinesterase. The enzyme is involved in neurotransmission; loss in signaling due to diazinon causes insect muscle to fail to function, eventually leading to death [2].
Uncontrolled use spreads of insecticide chemical contaminants into soil, water, and food and produces deleterious consequences on biodiversity, the environment, and human health. Diazinon is highly poisonous to beneficial insects (bees), birds [3], aquatic species [4], and other wildlife [5]. According to the International Agency for Research on Cancer (IARC) report, diazinon may cause cancer in humans [6]. In 2004, although the USA banned diazinon in residential areas, it is still legally available for agricultural applications. This presents a danger of contaminating soils [7], food [8], and water [3, 4] with diazinon, and it is an eminent element of threat to human health and biodiversity. Depending on the time and amount of exposure to diazinon, humans can have several neurological problems, such as dizziness, headache, weakness, difficulty breathing, or death [9]. Recent studies have shown that diazinon inhibits acetylcholinesterase [10], induces oxidative stress [11], promotes inflammation, and causes DNA damage [12]. Therefore, monitoring diazinon residue levels is essential for food and biosafety, human health, and environmental protection [5].
Early detection of toxic environmental and food contaminants such as insecticides is a challenge due to the small molecule nature of these chemicals [13, 14, 15]. At present, the most commonly used methods for insecticide analysis demand highly sophisticated machinery, heavy investment (or high maintenance cost), and the need for skilled workers to analyze the samples. Moreover, these methods are challenging to deploy in remote areas due to a lack of funds, bulkiness, and mobility issues. Examples of these methods include high-performance liquid chromatography (HPLC), mass spectrometry (MS), and gas chromatography, which demand laborious sample preparation [14]. Therefore, to overcome the challenges in the early detection of insecticides, a simple, quick, sensitive, specific, and rapid sensor assay is required [16, 17]. Aptamer sensors provide an attractive alternative in biowaste management via effective and efficient detection. Aptamers are single-stranded DNA or RNA (ssDNA or ssRNA) molecules that can selectively bind to specific targets [18]. Recently, aptameric sensors for detecting small molecules have drawn the intense attention of researchers and technologists as the method of choice because they meet several criteria that fulfill the demands for cheaper, quick, simple, easy, and sensitive detection [13, 19, 20]. For instance, Coonahan et al. [21] estimated the cost of aptamer-based assays to be as low as 0.25 USD/sample, while Trinh et al. [15] compared the cost of HPLC to be 80 to 100 times higher than that of aptasensors. Moreover, aptamers can be further developed as label-free and machine-independent sensing platforms [15, 21, 22].
Aptamers present several advantages over traditional methods of detection. It
can form a versatile three-dimensional structure that helps form a precise
binding pocket to recognize its cognate target [21]. Interactive binding via van
der Waals forces, hydrogen bonding, electrostatic interactions, stacking of flat
moieties, pi-pi interactions, pi-anionic interactions, steric hindrances, and
shape complementarity contribute to the specificity and high affinity. Aptamers
are isolated using a stringent selection protocol of the SELEX process [23]. The
principle of the SELEX method is based on the binding to targets of ssDNA or RNA
from a random pool (library of 10
Here, we report the development of novel ssDNA aptameric sensors to detect toxic food and the environmentally contaminating insecticide diazinon. We successfully identified three ssDNA candidate aptamers that bind to diazinon efficiently and effectively. The isolated aptamers displayed robustness in detecting diazinon even at a very low concentration of up to 148 nm. Moreover, DIAZ-03 could be differentiated from other related organophosphorus insecticides (fenthion, fenitrothion, and malathion). Next, we demonstrated its applicability in detecting diazinon from real samples of vegetables and fruit. We believe these aptamers can be custom designed as recognition elements in biosensing coupled with nanoparticles for bioremediation to remove contaminants [13]. The novel sensors present a promising future in bioprocessing, agriculture, food production, agri-waste treatment, and environmental protection. Overall, aptameric sensors could be pivotal in helping environmentalists and technologists meet several sustainable development goals (SDGs), including clean food, water, and a safe environment.
The chemicals diazinon, fenthion, fenitrothion, and malathion (HPLC purification
grade) were purchased from Sigma–Aldrich Inc. (St. Louis, MO, USA). All chemical
insecticides were dissolved in acetone and then diluted in 1x SELEX buffer (20 mM
HEPES, 1 M NaCl, 10 mM MgCl
The size of ssDNAs in the library used was 72 bp, which included the following:
N-30 region generated by randomly mixing the A-C-T-G bases and primer region
(forward and reverse): (1) Library: 5
A 500 pmol ssDNA library was mixed with 2500 pmol capture strand (1:5 ratio) in
250
Three aliquots of eluent after diazinon addition were concentrated by a 3 KDa
filter (EMD Millipore Amicon Ultra0.5 Centrifugal Filter Units, Millipore, cat.
No. 732-6204) and were used as the templates for large-scale PCR. The reverse
primer was replaced by a biotinylated reverse primer in this step. Four PCR tubes
containing 30
Purified DNA from the last round of library selection was used to clone the candidate aptamers. As mentioned earlier, PCR amplification of selected DNA was carried out, and cloning was performed using the T-Blunt™ PCR Cloning kit (SolGent, Daejeon, Korea) following the manufacturer’s instructions. A total of 96 individual colonies were randomly picked for plasmid extraction. Plasmid DNAs were extracted by using the HiGene™ Plasmid Mini Kit (Ver 2.0; BIOFACT, Daejeon, Korea) following the manufacturer’s instructions. All plasmids were sent for sequencing service by COSMO Tech Company (Seoul, Korea). The candidate aptamer sequences were analyzed and classified into groups using the Clustal Omega program [27] and aligned manually to remove spaces. Next, SELEX was performed with a few representative clones whose copy numbers were predominant in sequence data to confirm diazinon binding.
Sequences were analyzed to make a 2D structure using the Mfold program [28]. The
aptamer sequences that form a stem–loop structure with eight base-pair stems
were selected for the aptamer assay with fluorescein (FAM) conjugated to the
5
The quenching efficiency was determined by optimization of the ratio of the
sensor to the quencher. The 2x concentration of FAM-sensor 100 nM was mixed with
a 13-bp quencher by twofold serial dilution of range from 0 nM to 500 nM with an
equal volume (60
The K
The LOD for each sensor was calculated based on the formula CL = K*Sd/S [29], where Sd is the standard deviation of blank sample; K refers to a signal-to-noise ratio (S/N) (which was used in this study with 3.3); S is the slope of detecting linear between the fluorescence intensity enhancement versus low concentrations [29] of diazinon. The specificity of the sensors toward diazinon was estimated via cross-reactivity testing. Three organophosphate pesticides, fenthion, fenitrothion, and malathion, were used to compare the cross-reactivity of the selected aptamers.
The optimal conditions for detecting diazinon from vegetable and fruit tissue
extracts were evaluated using different pH values, temperatures, and incubation
times. The effect of pH was studied using all samples (sensor/quencher and
diazinon) by preparing in 1x SELEX buffer with different pH values (5.0, 6.0,
7.0, 7.5, and 8.0) incubating at room temperature for 40 min before measurement.
The effect of incubation time on all samples was studied by preparing samples in
1x SELEX buffer at pH 7.5 and measuring the signal at different time points from
0 min to 60 min. Finally, the effect of temperature was estimated by performing
the reaction in 1x SELEX buffer (pH 7.5 and 40 min) at various temperatures, such
as 20
To prepare plant tissue extracts, tomato, Chinese cabbage, and apples were
selected as examples to develop a biosensor assay. Chinese cabbage (cultivar: Wa
Wa Wa Sai), tomato (cultivar: Chal), and apple (cultivar: Fuji) were purchased
from TopMart (Jinju, Gyeongsangnam-do, Korea). Fifty grams of sample was first
cut into small pieces and homogenized with 50 mL of 20 mM Tris-HCl buffer (pH
7.5) by using a food blender and then centrifuged for 10 minutes at 4
The structure-switching SELEX process was employed [23] to identify ssDNA
aptamers specific to diazinon. This method was based on the principle of
structure switching and elution upon target binding [15, 18]. The random ssDNA
library (of
Design of oligonucleotide probes and monitoring of the SELEX
process. (A) An overview of a typical SELEX cycle to identify and isolate
specific ssDNA aptameric sequences. Random ssDNA libraries flanked by fixed
primer annealing sites for PCR amplification are incubated with the target
molecule, and aptamer-target complexes are separated from unbound ssDNA. PCR
amplification with the selected ssDNA followed by cloning, sequencing, and strand
separation for the next SELEX round. The SELEX cycle was repeated several times
to enrich the high-affinity binding ssDNA. (B) The random ssDNA library was
hybridized with a short capture strand via biotinylated complementary sequences,
wherein ‘N’ means any nucleotide generated by random mixing of A-T-G-C bases. (C)
Electrophoresis of eluent obtained after the 10th SELEX round (10
Confirmation and prediction of the secondary structures of candidate aptamers. (A) Stepwise illustration of aptamer selection—individual clones (plasmids), sequence amplification, strand separation, and confirmation of size and band intensity via electrophoresis, where NC, Negative control; Af. C, the mixture obtained after adding a column; W1, the first wash; W50, the 50th wash; Ex, the eluent after incubation with the target diazinon. (B) The secondary structure of three candidate aptamers was predicted using the Mfold program [28].
The selected PCR products were cloned, and numerous individual colonies were
obtained that carry plasmids with a particular PCR fragment representing each
candidate aptameric ssDNA. A total of 96 colonies were randomly picked for
sequencing. The sequences were subjected to bioinformatics analysis; sequence
alignment and similarity were measured. Each sequence contained an 18 bp forward
primer and 22 bp reverse primer at the 5
Aptamer ID | Sequence | Counts |
DIAZ-01 | 5 |
21 |
D1.1 | 5 |
2 |
D1.2 | 5 |
2 |
D1.3 | 5 |
1 |
D1.4 | 5 |
1 |
D1.5 | 5 |
1 |
DIAZ-02 | 5 |
13 |
D2.1 | 5 |
2 |
D2.2 | 5 |
1 |
D2.3 | 5 |
1 |
D2.4 | 5 |
1 |
D2.5 | 5 |
1 |
D2.6 | 5 |
1 |
D2.7 | 5 |
1 |
DIAZ-03 | 5 |
10 |
D3.1 | 5 |
1 |
D3.2 | 5 |
2 |
D3.3 | 5 |
2 |
The secondary structure of the newly discovered candidate aptameric sequences was predicted to examine the suitability and stability of the novel biosensor design (Fig. 2B). The sequences were analyzed by Mfold software [28]. The free energy of the sequences was compared, and all aptamers were found to have a short stem of 11 to 13 bp that could be used to form base pairing with a 13-bp dabcyl quencher.
The unique properties of ssDNA aptamers provide unparalleled opportunities to
develop novel biosensors that can specifically bind to target molecules and
produce a detectable signal. To obtain the fluorescence signal, the candidate
ssDNA aptamer was modified with fluorescein amidite (FAM) at the 5
Detection strategy and characterization of fluorescent aptamer
sensors against diazinon. (A) Graphic illustration of detection using a
strand-displacement-based fluorescence assay and quenching with a dabcyl-modified
quencher. Sensors are shown in blue line with 6-FAM (green circle) at the
5
Next, the binding affinity of aptameric sensors with diazinon was determined by
measuring the gain of fluorescence intensity (FL) by applying various
concentrations of the target molecule. DIAZ-01-F, DIAZ-02-F, and DIAZ-03-F were
incubated with quencher-strand in the optimal ratio that was determined by prior
quenching analysis. A total of 8 different concentrations of diazinon (0
To determine the specificity of the novel ssDNA aptameric sensors developed in this study, we tested the cross-reactivity of the DIAZ-F biosensors against three other organophosphate insecticides, fenthion, fenitrothion, and malathion (Fig. 4). The selected insecticides possess structural and physicochemical properties similar to those of diazinon’s specific target. Detailed analysis of specificity showed that both DIAZ-01-F and DIAZ-02-F had a relatively high level of cross-reactivity to fenthion and fenitrothion. The organophosphorus insecticides shared an aromatic ring structure (Fig. 4B–E). Notably, all three aptamers successfully discriminated between malathion and diazinon’s specific target. Moreover, malathion did not bind to any aptasensors. Among all aptamers, DIAZ-03F was found to be the most specific, as it did not bind to fenitrothion or malathion and showed weaker binding to fenthion insecticide. Altogether, the ssDNA aptamers exhibited high affinity and specific binding to the target molecule diazinon. However, DIAZ-1-F and DIAZ-2-F showed weaker binding to the nonspecific targets fenthion and fenitrothion due to the presence of core ring structures and phosphorothioate bonds. Overall, DIAZ-03-F performed best in the specific recognition of the target molecule.
Measurement of the cross-reactivity of aptamer sensors. Cross-reactivity analysis using three other insecticides: (A) diazinon; (B) fenthion; (C) fenitrothion; (D) malathion; and (E) chemical structure of insecticides.
Vegetables and fruits form a significant ingredient of food and nutrition and
are essential for a balanced human diet. This study assessed the
applicability of newly designed aptameric sensors to detect insecticide
contamination in food. The effect of pH, temperature, and incubation time was
investigated as food samples present complexities. The DIAZ-02-F sensor and
quencher mixture (1:5 ratios, i.e., 50 nM:250 nM) was incubated with 10
Analysis of diazinon concentration using the aptameric sensor in vegetable and fruit tissue extracts. (A) Determination of optimal conditions for diazinon detection using the DIAZ-02-F sensor. (B) Analysis of diazinon concentration using the DIAZ-02-F sensor in tissue extracts of vegetables/fruits—tomato, Chinese cabbage, and apple.
Altogether, the optimization experiments provided the best protocol to conduct
the experiments using the following conditions: 1x SELEX buffer (for sensor:
quencher mixture or food extract), pH 7.5, incubation at 30
In conclusion, we report the discovery of three novel ssDNA aptameric sensors
that bind to diazinon with a low K
MHTC—Methodology, Validation, Formal analysis, Writing—original draft preparation, and Data curation. USK—Conceptualization, Methodology, Validation, Formal analysis, writing—original draft preparation, Formal analysis, writing—original draft preparation, writing—review and editing. KHT—Validation. YC—Methodology, HL—Validation. YK—Visualization. SK—Validation. CHK—Methodology. SHK—Visualization. WSC—Resources, Writing—review and editing. SYL—Resources, Writing—review and editing. JCH—Conceptualization, Funding acquisition, Resources, Supervision, Formal analysis, writing—original draft preparation, Writing—review and editing. All authors have read and agreed to the published version of the manuscript.
Not applicable.
Authors would like to express the gratitude to all other lab members who helped during experiments.
This research was supported by funding from the BioGreen21 Agri-Tech Innovation Program (Project PJ01623501), Rural Development Administration, Republic of Korea, and Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2020R1A6A1A03044344 and 2020R1F1A1074027).
The authors declare no conflict of interest.