A protocol for the in vitro selection and characterization of group-specific phthalic acid ester- binding DNA aptamers is presented. The application of the selected aptamer in an electrochemical aptasensor is also included.
Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the rapid growing of congeners. DNA aptamers have been increasingly applied as recognition elements on biosensor platforms, but selecting aptamers toward highly hydrophobic small molecule targets, such as PAEs, is rarely reported. This work describes a bead-based method designed to select group-specific DNA aptamers to PAEs. The amino group functionalized dibutyl phthalate (DBP-NH2) as the anchor target was synthesized and immobilized on the epoxy-activated agarose beads, allowing the display of the phthalic ester group at the surface of the immobilization matrix, and therefore the selection of the group-specific binders. We determined the dissociation constants of the aptamer candidates by quantitative polymerization chain reaction coupled with magnetic separation. The relative affinities and selectivity of the aptamers to other PAEs were determined by the competitive assays, where the aptamer candidates were pre-bounded to the DBP-NH2 attached magnetic beads and released to the supernatant upon incubation with the tested PAEs or other potential interfering substances. The competitive assay was applied because it provided a facile affinity comparison among PAEs that had no functional groups for surface immobilization. Finally, we demonstrated the fabrication of an electrochemical aptasensor and used it for ultrasensitive and selective detection of bis(2-ethylhexyl) phthalate. This protocol provides insights for the aptamer discovery of other hydrophobic small molecules.
Along with rapid economic development, acceleration of industrialization, and urban construction, environmental pollution is more severe than ever. Typical environmental pollutants include heavy metal ions, toxins, antibiotics, pesticides, endocrine disruptors, and persistent organic pollutants (POPs). Besides metal ions and toxins, other pollutants are small molecules that quite often consist of a variety of congeners. For example, the most toxic POPs include polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), polybrominated biphenyl ethers (PBDEs), polychlorinated dibenzo-p-dioxin (PCDDs), polychlorinated dibenzofuran (PCDFs), and phthalic acid esters (PAEs)1,2, which all consist of many congeners. Small molecule detection has been mainly performed by chromatography/mass spectrometry-based techniques due to their diversity of applications3,4,5,6. For on-site detections, antibody-based methods have recently been developed7,8,9. However, since these methods are highly specific for a certain congener, multiple tests must be performed. What is more serious is that the novel congeners grow so fast that their antibodies can't be generated in time. Therefore, the development of group-specific biosensors to monitor the total levels of all congeners in one test may provide an invaluable metric for assessing environmental pollution status.
Recently, nucleic acid aptamers have been widely applied as recognition elements in various biosensing platforms due to their capability of recognizing a wide variety of targets, from ions and small molecules to proteins and cells10,11,12. Aptamers are identified through an in vitro method called systematic evolution of ligands by exponential enrichment (SELEX)13,14. SELEX begins with the random synthetic single strand oligonucleotide library, which contains approximately 1014-1015 sequences. The size of the random library ensures the diversity of the RNA or DNA candidate structures. The typical SELEX process consists of multiple rounds of enrichment until the library is enriched in sequences with high affinity and specificity to the target. The final enriched pool is then sequenced, and the dissociation constants (Kd) and selectivity against potential interfering substances are determined by different techniques such as filter binding, affinity chromatography, surface plasmon resonance (SPR), etc.15
Due to the extremely poor water solubility and lack of functional groups for surface immobilization, the aptamer selection of POPs is theoretically difficult. Significant advances for SELEX have speeded up the discovery of aptamers. However, the selection of group-specific aptamers for POPs has not yet been reported. So far, only PCB-binding DNA aptamers with high specificity for a certain congener have been identified16. PAEs are mainly used in polyvinyl chloride materials, changing polyvinyl chloride from a hard plastic to an elastic plastic, thus acting as a plasticizer. Some PAEs have been identified as endocrine disruptors, can cause serious damage to liver and kidney function, reduce the motility of male sperm, and may result in abnormal sperm morphology and testicular cancer17. Neither the compound- nor group-specific PAE-binding aptamers have been reported.
The goal of this work is to provide a representative protocol for selecting group-specific DNA aptamers to highly hydrophobic small molecules such as PAEs, a representative group of POPs. We also demonstrate the application of the selected aptamer for environmental pollution detection. This protocol provides guidance and insights for the aptamer discovery of other hydrophobic small molecules.
1. Library and Primer Design and Synthesis
2. Synthesize the Anchor Target and Its Immobilization onto Epoxy-Activated Agarose Bead
3. SELEX
4. High-Throughput Sequencing
5. Kd Determination of Selected Aptamer Candidates using Magnetic Bead-Based Quantitative PCR (qPCR)
6. Relative Affinity and Specificity Test by Competitive Assays
7. Fabrication and Electrochemical Measurements of DEHP Electrochemical Biosensors
We designed and synthesized the amino group functionalized dibutyl phthalate (DBP-NH2) as the anchor target (Figure 1F). We then performed the DNA aptamer selection of PAEs using DBP-NH2 as the anchor target and following the classical target immobilization-based method (Figure 2). In each round, a pilot PCR was performed using the denatured PAGE to optimize the cycle number of PCR (Figure 3). The denatured PAGE, instead of native PAGE, is strongly suggested because we and other colleagues have found that the suspected high molecular weight by-products shown on the native gels at the higher cycle numbers are actually crosslinking complexes formed by sequences of the right size. Thus, the band intensity of the denatured PAGE can truly reflect the amount of the right product. The single stranded DNA generation is another critical step, and many methods for this step have been reported28. The λ exonuclease digestion method is the cheapest method. The success of the single-stranded DNA generation can be conveniently checked by native PAGE, where the PCR product is shown as single (first several rounds of SELEX) or multiple bands, while only one band is shown after the digestion (Figure 4).
The SELEX was stopped after the fifth round of the selection because of the significant quantity of DNA accumulated at the top of the PAGE, even after the denaturation treatment (wherein DNAs are heated at 95 °C for 10 min in 2 × TBE–containing 8.3 M urea), which suggested serious cross-linking between DNAs and also indicated that the sequences were enriched. Therefore, the pool4 was sent out for the high-throughput sequencing. The high-throughput result showed that the top 100 most frequently occurring sequences were highly conserved and were from one family. The top sequence DBP-1 displayed nanomolar affinity (Figure 5) and good group-specificity to PAE congeners (DBP, BBP, DEHP) (Figure 6) via the convenient qPCR assays, as described in the protocol section.
DBP-1 has been used to construct a strand displacement-based electrochemical aptasenor accordingly to our previously reported work29. The DEHP sensor can sensitively and selectively respond to DEHP (Figure 7). The common environmental pollutants such as heavy metal ions, antibiotics, and small molecules with similar functional groups showed very weak response to DBP-1.
Figure 1: Chemical structures of (A) PAEs, (B) BBP, (C) DBP, (D) DEHP, (E) 4-OH-DEHP, (F) DBP−NH2. This figure has been reprinted with permission from Han Y. et al.22 Please click here to view a larger version of this figure.
Figure 2: Group-specific DNA aptamer selection procedure for PAEs using DBP-NH2 as an anchor target. This figure has been reprinted with permission from Han Y. et al.22 Please click here to view a larger version of this figure.
Figure 3: Representative results from PCR cycle optimization using denatured PAGE. From left to right, the lanes represent: 20 bp DNA ladder standard, 15 cycles (no DNA template), 20 cycles (no DNA template), 25 cycles (no DNA template), 30 cycles (no DNA template), 15 cycles, 20 cycles, 25 cycles, and 30 cycles. Optimal conditions were observed at 25 cycles, where the single product band was at high intensity without unwanted by-products, furthermore, no band was observed in the corresponding blank control. Please click here to view a larger version of this figure.
Figure 4: The optimization of λ exonuclease reaction to digest the phosphorylated strand using native PAGE. From left to right, the lanes represent: 20 bp DNA ladder standard, negative (no λ exonuclease), 2U, 5U, 8U, and 10U. The minimum amount of enzyme was observed at 2U, where the double PCR product becomes single-stranded DNAs. Please click here to view a larger version of this figure.
Figure 5: Affinity measurements of DBP-1 by qPCR-based assays. The error (standard deviation, SD) of the Kd 's was calculated from three measurements of the same sample. This figure has been reprinted with permission from Han Y. et al.22
Figure 6: Relative affinity measurements of DBP-1 for free DBP-NH2 (10 µM), DBP (10 µM), DEHP (10 µM), BBP (10 µM), ethyl acetate (10 µM), benzoic acid (10 µM), phthalic acid (10 µM), and other via competition assays. Other: a mixture of potential interferences (glucose, kanamycin, ampicillin, and ethanol) all at 10 µM. The ratio (the relative affinity) was calculated by dividing the number of aptamers released from the beads in the presence of the sample by the number of aptamers released from the beads in the PAE binding buffer. The bars represent mean ± SD. The SDs were calculated from three individual measurements. Please click here to view a larger version of this figure.
Figure 7: DEHP electrochemical biosensor for ultrasensitive and ultraselective detection of DEHP: mechanism (A), SWV curves (B), calibration curve (C), and selectivity tests (D). The errors (SDs) were calculated from three individual measurements. This figure has been reprinted with permission from Han Y. et al.22 Please click here to view a larger version of this figure.
One outstanding benefit of aptamers is that they are identified through the in vitro method SELEX, while antibodies are generated via in vivo immunoreactions. Therefore, aptamers can be selected with desired target specificity under well-designed experimental conditions, whereas antibodies are limited to physiological conditions.
To facilitate the separation of bound sequences from free sequences, several modified SELEX have recently been reported, in which capillary electrophoresis30, microfluidics31, magnetic / acrylic beads / agarose beads14, etc., have replaced nitrocellulose filters or affinity columns to achieve more efficient separation. Among these techniques, the bead-based methods have been most widely used for the aptamer selection of small molecule targets due to their simple set-up, easy operation, and being amenable to small molecule analyses.
There are two groups of methods that are commonly used for aptamer selection of small molecule targets: the target13 and library14 immobilization-based methods. In the former group of methods, the small molecule targets are immobilized on the solid phase such as functionalized magnetic beads or agarose beads via covalent coupling reactions. Libraries are incubated with small-molecule coated beads, and those sequences that do not bind or bind weakly to the target on the solid phase are removed simply by performing washing and centrifugation or magnetic separation steps. The bound sequences are subsequently eluted and amplified by PCR. The small molecule targets must have at least one functional group available for the coupling reaction. For those without suitable functional groups, the functional group has to be incorporated into the original target through organic synthesis. The synthesis of the carefully designed target could involve multiple steps, and sometimes is quite challenging as well. The structural modification of the targets could also strongly affect the binding sites and affinity with their aptamers. To avoid these problems, library immobilization-based methods have been developed, where the library is hybridized to complementary DNA capture probes on magnetic beads, and the binding sequences fall off the beads upon binding with the targets. In this group of methods, the targets are added in the binding buffer and no functional groups are required. PAEs are the ester derivatives of phthalate acid, and a typical PAE consists of a phthalate acid ester group and one or two alkyl chains (Figure 1A-D). PAEs have no functional groups available for solid phase immobilization. Thus, the library immobilization-based methods seem more attractive for the aptamer selection of PAEs.
A well-controlled dispersion status is critical to ensure the desired enrichment of the library via SELEX. However, PAEs are extremely hydrophobic and easily form aggregates in aqueous solutions at a concentration higher than several µM, even in the presence of multiple surfactants to facilitate dispersion. Thus, the dispersion status of PAEs is hard to control, and it would be difficult to select aptamers that specifically bind to individual PAE molecules. To solve the solubility problem, target-immobilization methods were chosen, in which PAEs were immobilized on the hydrophilic beads via covalent bonding. By using this immobilization strategy, the targets should be dominantly present on the bead surface in a single molecular state, instead of aggregates.
The structural design of the anchor target is also quite critical for the successful selection of group-specific aptamers. Three factors need to be considered for the structural design. The first factor is the purpose of the aptamer selection. Considering the purpose of selecting group-specific aptamers, it is essential to expose the common group of PAEs for aptamer binding and prevent the rest of the parts from participating in the aptamer binding. Thus, the functional group should be introduced on the terminal of the side chain. At the beginning, we designed OH-functionalized DEHP (4-OH−DEHP) (Figure 1E) as the anchor target and immobilized it on the carboxylic acid magnetic beads16. Thus, the alkyl chains were exposed on the surface of the matrix. We tried to choose magnetic beads with carboxyl groups as the solid matrix. No obvious affinity improvement was observed after five rounds of selection, and the nonspecific absorption onthe bare matrix was kept strong.
Therefore, DBP-NH2 was later designed based on the following thoughts: (1)The phthalate group is more likely to interact specifically with the aptamer through the π-π stack and the hydrogen bond than the alkyl chain; (2) NHS-mediated carbodiimide reaction is mild and has a very efficient coupling efficiency, so -NH2 is introduced at the end of an alkyl chain of DBP; (3) It is easy to operate with the magnetic beads as a solid phase partition, but the nonspecific adsorption of the library is strong. Although the loss of the agarose beads during separation is higher than the magnetic beads, the nonspecific adsorption of the library is much lower. Thus, DBP-NH2 was immobilized on the epoxy-activated agarose beads, and the phthalic acidester group was exposed on the surface of the matrix.
The choice of selection buffer is important, especially for small molecule targets with diverse solubility. The binding buffer needs to be carefully prepared to avoid aggregation and ensure a good dispersion state of POPs during the whole aptamer selection and characterization process. In our study, we found that DEHP can't dissolve in regular buffers without surfactants, showing two distinct layers. The layer disappears upon addition of multiple surfactants in the optimized quantity. The clear solutions need to be stored at temperatures higher than 20 °C to maintain its status. Details are provided in protocol step 3.1.
Single-stranded DNA generation from double-stranded PCR products is one critical step in the SELEX process. Several different methods have currently been described in the literature32,33, including asymmetric PCR, λ exonuclease digestion, magnetic separation with streptavidin-coated beads, and size separation by denaturing urea-polyacrylamide gel.
Different methods have their own strengths and weaknesses. Currently, the most commonly used method for the generation of ssDNA is magnetic separation with streptavidin-coated beads. The outstanding advantage of this method is its time-saving and simple operation. The drawback is its high cost compared with other methods. In contrast, the size separation-based method, using reverse primers with GC-rich stem–loop structure, is one of the cheapest methods, while the yield of single-stranded DNA is the lowest among these methods32. In this protocol, we described the use of λ exonuclease digestion to generate single-stranded DNA, which is one of the cheapest methods. We found that the yield of single-stranded DNA is comparable with the two methods of magnetic separation and streptavidin-coated beads. Furthermore, we found that the exonuclease reaction was inhibited by the high concentration of salt. The incomplete digestion of the PCR product reported in the literature33 was likely due to too much salt existing in the PCR product. In addition, the λ exonuclease is highly active and inexpensive (Figure 4).
The characterization and validation of aptamers is laborious, time-consuming, and embodies a major bottleneck in the aptamer discovery pipeline33. Most techniques are mass-sensitive methods which work well for the larger aptamer binding partners (>10,000 amu), but are not sufficiently sensitive to measure the interactions with low molecular weight targets (<1,000 amu)15. The characterization and validation of aptamers of hydrophobic small molecules like PAEs is even more difficult. Their poor water solubility results in the unsaturation of the titration curve, which prevents the Kd's determination in solution or immobilizes aptamers on surface. Therefore, we determined the Kds of the identified aptamers by high throughput sequencing by immobilizing DBP-NH2 on the hydrophilic magnetic beads to avoid the solubility problem. The relative affinities and selectivity of the aptamers to other PAEs were then determined by the competitive assays, where the aptamer candidates were pre-bounded to the DBP-NH2 attached magnetic beads and released to the supernatant upon incubation with the tested PAEs or other potentially interfering substances. The competitive assay was applied because it provided a facile affinity comparison among PAEs that had no functional groups for surface immobilization. In addition, magnetic bead-based fluorescent assays are suitable for the affinity study of small molecule-aptamer interactions34. However, we found that the magnetic beads sometimes cause fluorescence quenching for unknown reasons. Thus, the qPCR assays were used for the affinity measurements.
One critical technique tip for the electrochemical biosensor described in this study is the surface passivation of the electrode35. Due to the high hydrophobicity of DEHP, it has a strong tendency to be nonspecifically absorbed onto the gold electrode, leading to the failure of the detection. The most commonly used surface passivation agent, 6-mercapto-1-hexanol (MCH)36,37, is not sufficient to prevent the nonspecific absorption of DEHP, while we found that HS-(CH2)2-[OCH2CH2]6-OCH3 was effective enough to enable the sensitive detection of DEHP38.
This procedure describes a protocol for selecting group-specific DNA aptamers of highly hydrophobic small molecules and an application of the selected aptamer in an electrochemical biosensor. The protocol helps with the selection of other hydrophobic small molecules and provides insights on sensor development of highly hydrophobic small molecules as well. The aptamer selection process belongs to the category of target immobilization-based methods. The limitations of this type of method also exist for this protocol, for example the need for complicated synthesis of anchor targets and the impacts of the solid phase on aptamer binding. The attractive advantages of the electrochemical biosensors described in this protocol include their simple design and high sensitivity. The major drawback is their limited precision due to their extremely broad dynamic range. Therefore, the biosensors described here are more suitable for screening tests, instead of quantitative measurements of the targets.
The authors have nothing to disclose.
We are grateful for financial support from the National Natural Science Foundation (21675112), Key project of science and technology plan of Beijing Education Commission (KZ201710028027) and Yanjing Young Scholar Program of Capital Normal University.
UV-2550 | Shimadzu,Japan | protocol, section 3.8.2 | |
DNA Engnine Thermal cycler,PTC0200 | BIO-RAD | section 3.5.1.2 and 3.5.2 | |
C1000 Touch | BIO-RAD | section 5.3.6 and 6.3 | |
VMP3 multichannel potentiostat | Bio-Logic Science, Claix, France | section 7.4,7.8 and 7.11 | |
Epoxy-activated Sepharose 6B | GE Healthcare (Piscataway, NJ, USA) | 10220020 | argarose beads, section 2.3 and 3.3 |
Dynabeads M-270 carboxylic acid magnetic beads | Invitrogen, USA | 420420 | magnetic beads,section 5.2. and 5.3 |
Premix Taq Hot Start Version | Takara,Dalian,China | R028A | polymerase, section 3.5.1.1 |
PARAFILM Sealing Membrane | Bemis, USA | PM-996 | section 3.6.5 |
Lambda Exonuclease | Invitrogen, USA | EN0561 | section3.7.1.2.The 10 × reaction buffer is provided along with λ exonuclease by the provider. |
Dr. GenTLE Precipitation Carrier |
Takara,Dalian,China | 9094 | section 3.6.2 and 3.8.1 |
UNIQ-10 PAGE DNA recovery kit | Sangon Biotech (Shanghai) | B511135 | section 4.2 |
SYBR Gold nucleic acid gel stain | Invitrogen, USA | 1811838 | nucelic acid stain dye, section 3.5.1.5 |
SYBR Premix Ex Taq II | Takara,Dalian,China | RR820A | polymerase mix contaning polymerase and dNTPs, section 5.3.5 |
2-(N-Morpholino)ethanesulfonic acid (MES) | Sigma-Aldrich | CAS: 1132-61-2 | section 5.2.1 |
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) | Invitrogen, USA | CAS: 25952-53-8 | section 5.2.2 |
N-hydroxysuccinimide (NHS) | Sigma-Aldrich | 6066-82-6 | section 5.2.3 |
mercaptohexanol (MCH) | Sigma-Aldrich | CAS: 1633-78-9 | section 7.7 |
Gold electrode | Shanghai Chenhua | CHI101 | section 7.4. – 7.11 |
tris(2-carboxyethyl) phosphine hydrochloride (TCEP) | Sigma-Aldrich | CAS: 51805-45-9 | section 7.5 |
O-(2-Mercaptoethyl)-O'-methyl-hexa-(ethylene glycol) | Sigma-Aldrich | CAS: 651042-82-9 | section 7.7 |
diethylhexyl phthalate (DEHP) | National Institute of Metrology, China | CAS: 117-81-7 | section 7.11 |
Tween 20 | Sigma-Aldrich | CAS: 9005-64-5 | polyoxyethy-lene(20) sorbaitan monolaurate |
Triton X-100 | Sigma-Aldrich | CAS: 9002-93-1 | non-ionic surface active agent |
PBS | Sigma-Aldrich | P5368 | 10 mM phosphate buffer containing 1 M NaCl, pH 7.4 |