Aptamer Structure Switch Coupled with Horseradish Peroxidase

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Aptamer Structure Switch Coupled with Horseradish Peroxidase Labeling on Microplate for Sensitive Detection of Small Molecules Yapiao Li, Linlin Sun, and Qiang Zhao Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05606 • Publication Date (Web): 24 Jan 2019 Downloaded from http://pubs.acs.org on January 24, 2019

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Analytical Chemistry

Aptamer Structure Switch Coupled with Horseradish Peroxidase Labeling on Microplate for Sensitive Detection of Small Molecules

Yapiao Li,1,2 Linlin Sun1,2, Qiang Zhao1,2*

1. State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China 2. University of Chinese Academy of Sciences, Beijing 100049, China * Corresponding author E-mail: [email protected] Tel: +86-10-62849892. Fax: +86-10-62849892.

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Analytical Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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Abstract Detection of small molecules with good sensitivity, high throughput, simplicity, and generality using aptamers is desired, but still remains challenging. We described an aptamer structure switch assay coupled with horseradish peroxidase (HRP) labeling on microplates for sensitive absorbance and chemiluminescence detection of small molecules. This assay relied on the competition between small molecule targets and the immobilized short complementary DNA of aptamer (cDNA) on a microplate in the affinity binding to a limited HRP-labeled aptamer. In the absence of targets, the HRP-labeled aptamer hybridized with the cDNA on the microplate, and HRP catalyzed substrate into product, generating absorbance or chemiluminescence signals. The binding of small molecule targets to aptamers caused displacement of HRP-labeled aptamers from the cDNA and signal decrease. In the chemiluminescence analysis mode, the assay achieved detection of aflatoxin B1 (AFB1), ochratoxin A (OTA) and adenosine triphosphate (ATP) with detection limits of 10 pM, 20 pM and 20 nM, respectively. This assay does not require enzyme-labeled small molecules or conjugating small molecules on solid phase. HRP as enzyme labeling here is ease of obtaining and highly active for signal amplification. This microplate assay is rapid and promising for high throughput analysis. It shows potential for wide application in the detection of small molecules.

Keywords: Aptamer, Small molecule, Competitive assay, Enzyme label, Absorbance, Chemiluminescence

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Sensitive detection of small molecules (e.g., metabolites, therapeutics, pollutants, and natural toxins, and etc.) is important and demanded in broad fields. As attractive nucleic acid affinity ligands with good selectivity and affinity,1,2 many aptamers have been available for a variety of small molecules now.3,4 Aptamer-based methods for small molecule detection are drawing wide and increasing interests in sensors and assays due to advantages of aptamers, such as easy chemical synthesis, low cost, facile modification for labeling and immobilization, good thermostability, and binding-induced structure switch property.5,6 These methods have included fluorescence assays, electrochemistry assays, and colorimetric methods, and etc.3-9 Enzymatic signal amplification is often used to enhance the detection sensitivity.5,10 Competitive enzyme linked aptamer assay (ELAA) is one of sensitive strategies for small molecule detection.10-15 It usually requires conjugation of small molecules on solid support and enzyme labeled aptamer, or it needs enzyme labeled small molecules and immobilized aptamer.10-15 The conjugation of small molecules on proteins (e.g., bovine serum albumin), enzymes, or solid surface is relatively difficult, challenging, and time consuming. The conjugation may disrupt the affinity binding between the small target and aptamer because the conjugation site may overlap with the binding site or interfere with the affinity binding. In addition, some small molecules lack of functional groups for labeling or immobilization. The sandwich like ELAAs using split aptamers or "kissing complex" rely on the assembly of oligonucleotides in the presence of targets, 16,17 and only work for limited aptamers that have specific secondary structures. To overcome the limitations of previous enzyme linked aptamer assays for small molecules, herein we describe a simple and general method for sensitive absorbance and chemiluminescence detection of small molecule targets on microplate by using target-binding induced aptamer structure switch assay coupled with horseradish peroxidase (HRP) labeling. It has been known that the aptamer-target binding is competitive with the hybridization of aptamer and its complementary sequences, 18-20 which is a unique feature for aptamer-based assays.5,19 Integrating aptamer structure switch and enzyme labeling can improve detection of targets.21-25 Invertase conjugate 3



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has been used as a label combining magnetic separation for detection of small molecules with a portable glucose meters.21-23 This approach is promising for tests in point-of-cares and in the fields by converting target detection into glucose detection, but it is relatively weak in sensitivity and high throughput analysis.21-23 Compared to other enzyme labels,21,24,25 HRP is ease of obtaining, shows good enzymatic activity, and enables high signal amplification. The use of HRP coupling with aptamer structure switch on microplate for optical analysis of small molecules is advantageous but still limited. In our strategy here, we propose a simple detection system using a HRP-labeled aptamer and a microplate coated with short complementary DNA (cDNA) of the aptamer for small molecule analysis. In the absence of small molecule targets, the HRP-labeled aptamer hybridizes with the cDNA on the microplate. HRP then initiates enzyme reaction to generate absorbance or chemiluminescence signals. When small molecule targets exist, the HRP-labeled aptamer binds to targets instead of the cDNA on microplate, causing signal decreases. With this strategy, we successfully detected three different small molecules of food safety and health interest with high sensitivity, including aflatoxin B1 (AFB1), ochratoxin A (OTA) and adenosine triphosphate (ATP).

EXPERIMENTAL SECTION Materials and Reagents All the DNA oligonucleotides (Table S1 in Supporting Information (SI)) were synthesized and purified by Sangon Biotech (Shanghai, China). The oligonucleotides had one biotin label at the 3’ terminal with a TEG (tetraethyleneglycol) linker. Other reagents and materials used in the experiment were shown in SI. Absorbance analysis and chemiluminescence analysis were conducted by a plate reader (SynergyTM H1, Biotek, USA).

Absorbance and Chemiluminescence Assays for Targets The cDNA coated microplate was prepared by the procedure described in SI. The biotinylated aptamer and the HRP-conjugated streptavidin at a ratio of 1 to 1 were 4


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incubated in the binding buffers (SI Table S2) at 4 °C for 30 min to achieve the HRP-labeled aptamer probe. In the assay, certain concentrations of target were mixed with HRP-labeled aptamer (1 nM) in the binding buffer and incubated for 10 min at 4 °C. After that, 100 μL of the mixture solution was transferred to the cDNA coated wells, and incubated for 30 min (for AFB1 detection) or 15 min (for OTA and ATP detection) at 4 °C. After washing three times with 200 μL of binding buffer, 100 μL of substrate solution was added to the wells. In the assays using absorbance analysis, 10-min incubation of TMB substrate solution was applied at room temperature, and then 100 μL of 1 M HCl was added to stop the reaction before measuring the absorbance at the wavelength of 450 nm by the plate reader. In the assays using chemiluminescence analysis, 8-min incubation of chemiluminescent substrate solution was applied at room temperature, and then chemiluminescence was immediately measured by the plate reader. Duplicate samples were tested. Each sample was measured three times, and the average value was used for data processing.

RESULTS AND DISCUSSION

Principle of Aptamer Structure Switch Assay Coupled with HRP Labeling on Microplate for Small Molecule Detection Figure 1 shows the principle of aptamer structure switch assay coupled with HRP labeling on microplate for small molecule detection. A cDNA of the aptamer is immobilized on the surface of a microplate. The sample solution containing a limited amount of HRP-labeled aptamer is added into cDNA coated wells. When small molecule target is absent, the HRP-labeled aptamer hybridizes with the cDNA on the microplate surface. As a reporter, HRP catalyzes substrate into product, generating high signals in absorbance or chemiluminescence. When small molecule target exists in the sample solution, the HRP-labeled aptamer binds with the target, not hybridizing with the cDNA on the microplate. With increase of targets, the HRP attached on the microplate decreases, leading to signal reduction. Finally, a rapid detection of small molecule targets is achieved by measuring signal changes through absorbance or 5



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chemiluminescence analysis with a plate reader.

Assay for AFB1 Detection To show the proof of principle of our proposed strategy, firstly we applied this strategy to detect aflatoxin B1 (AFB1), which is one of the most toxic substances among aflatoxins produced by Aspergillus flavus and Aspergillus parasiticus.26-28 AFB1 has been classified as Group 1 carcinogen by the International Agency for Research in Cancer.26-28 Many efforts have been made to develop assays for AFB1, and rapid and sensitive assays are highly demanded.26-28 We tested the feasibility of our method for AFB1 detection by using chromogenic substrate and absorbance analysis. We labeled HRP on the anti-AFB1 aptamer (Apt-AFB, SI Table S1) to obtain a HRP-labeled aptamer probe.29,30 We attached the cDNA (C14-AFB) of the aptamer on the microplate (SI Table S1). When AFB1 was not present, the HRP-labeled aptamer hybridized with the cDNA on the microplate, generating a high absorbance signal (Figure 2). When 10 nM AFB1 existed in the sample solution, the signal significantly decreased. It shows our strategy is feasible for the detection of AFB1. The length of cDNA had large effect on the assay performance, which is essential to the hybridization between cDNA and aptamer.18,19,30 We tested the cDNA with lengths ranging from 11 to 16 nucleotides (Figure 2 and SI Table S1). The absorbance signals of blank samples in the assay increased with the increase of lengths of cDNA, showing the interaction between cDNA and aptamer is enhanced by longer cDNA. The signal decrease caused by AFB1-binding increased with the increase of lengths of cDNA ranging from 11 to 14 nucleotides, and reached large signal decrease when the nucleotides were longer than 13 nucleotides. When the cDNA containing 14 nucleotides (C14-AFB) was applied, the percentage of signal decrease caused by AFB1 was higher (SI Figure S1). Thus, the cDNA C14-AFB was chosen in the assay for AFB1 detection. The concentration of MgCl2 in the binding buffer significantly affected the signals from the blank sample and the sample containing 10 nM AFB1 (SI Figure S2) 6


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because MgCl2 not only facilitates the hybridization of cDNA and aptamer, but also is important for the affinity binding between AFB1 and aptamer. Relatively larger percentage of signal decrease caused by AFB1 was obtained when the concentration of MgCl2 was 10 mM or higher (SI Figure S2B). To reduce the possible nonspecific adsorption in solution with low ionic strength, we finally chose the binding buffer of 10 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 50 mM NaCl and 1 mg/mL BSA for further experiments. Under optimal conditions, we achieved successful detection of AFB1 using the cDNA C14-AFB coated microplate and the HRP-labeled aptamer (1 nM). As shown in Figure 3A, the absorbance signals gradually decreased with the addition of AFB1. The assay using absorbance analysis allowed for the detection of AFB1 in the range from 0.2 nM to 2000 nM. The detection limit was 0.2 nM (~0.06 μg/kg). The maximum percentage of signal decrease caused by AFB1 was about 84%. We further lowered the detection limit of AFB1 to 10 pM (~0.003 μg/kg) by utilizing chemiluminescent substrate and a more sensitive chemiluminescence analysis in the assays (Figure 3B). The dynamic detection range covered about five orders of magnitude in the chemiluminescence analysis. The sensitivity of our assay for AFB1 is higher than some reported assays for AFB1 (SI Table S3).28 The detection limit of our assays is lower than the maximum regulation levels of AFB1 in food products set by European Union (2 μg/kg) and World Health Organization (WHO) (5 μg/kg).26 To test the selectivity of this method for AFB1 detection, we detected other mycotoxins including OTA, OTB, FB1, FB2, and ZAE. These tested substances did not cause significant absorbance decrease (SI Figure S3). The result shows that the assay is specific for the detection of AFB1. To assess the applicability of the assay for detecting AFB1 in complex sample matrix, AFB1 spiked in 20-fold diluted liqueur was tested by the assay utilizing absorbance analysis or chemiluminescence analysis (SI Figure S4). The obtained absorbance responses and chemiluminescence responses were similar to that from the binding buffer solution, and detection limits of absorbance and chemiluminescence analysis in the 20-fold diluted liqueur reached 0.2 nM and 20 pM, respectively. Our 7



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assay also allowed the detection of AFB1 spiked in maize flour samples with recovery rates ranging from 98% to 120% (SI Table S4).

Generality of Assays for Small Molecule Detection To test whether our method was a general approach for detection of small molecules, we further applied our strategy to detect ochratoxin A (OTA). OTA is produced by Aspergillus and Penicillium fungi, and OTA contamination often occurs in a variety of agricultural commodities and food.31,32 Our assay used an aptamer specific to OTA (Apt-OTA, SI Table S1).33 After testing different lengths of cDNA, we found the cDNA C7-OTA having 7 nucleotides complementary with the aptamer was preferred to give higher signal change upon OTA addition (SI Figure S5A). By using the cDNA C7-OTA coated microplate, sensitive detection of OTA was achieved. The detection limit of OTA was 1 nM (~0.4 μg/kg) in the assay using absorbance analysis (SI Figure S5B). The detection limit reached 20 pM (~0.008 μg/kg) by the assay using chemiluminescence analysis (Figure 4A), which is lower than that of many reported assays (SI Table S3).32 This method was selective for OTA (SI Figure S6). OTA spiked in 20-fold diluted liqueur samples could be detected in the assay, with detection limits at 1 nM and 20 pM for utilizing absorbance analysis and chemiluminescence analysis, respectively (SI Figure S7). The OTA detection limit of our method is lower than the permissible limit of OTA in some food products set by European Union (2-5 μg/kg).32 Our strategy also worked for detection of adenosine triphosphate (ATP), an important compound participating in many processes in living organism.8 An aptamer for ATP (Apt-ATP, SI Table S1) was applied.34 The cDNA C8-ATP of this aptamer was chosen for generating higher signal changes (SI Figure S8A). The detection limit of ATP in the binding buffer was 500 nM (Figure 4B and SI Figure S8B) for the assay using absorbance analysis. The assay using chemiluminescence analysis allowed for detection limit of 20 nM ATP (SI Figure S8C), lower than that of some reported aptamer assays (SI Table S3).7 This method showed good selectivity to ATP (SI Figure S9). The detection of ATP spiked in diluted serum sample was also achieved 8


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with a detection limit of 500 nM using absorbance analysis mode (Figure 4B). The successful detections of AFB1, OTA, and ATP show our strategy is general for detection of different small molecules. The different sensitivities of assays for AFB1, OTA, and ATP result from the different binding affinities of the aptamers. The aptamers for AFB1 and OTA have stronger binding affinity, and the reported dissociation constant (Kd) is around tens nM level.29,33 The aptamer against ATP has a Kd about 10 μM.34 The employment of aptamer having higher binding affinity allows for a lower detection limit. The use of HRP as enzyme labeling and chemilunmiscence analysis in the assay significantly amplifies the signals, and enables the detection limit much lower than the Kd of aptamers.

CONCLUSIONS In summary, we have developed an aptamer structure switch assay coupled with HRP labeling for small molecules by using complementary DNA coated microplate and HRP-labeled aptamer. Taking advantage of high enzymatic activity of HRP, the assay shows high sensitivity due to signal amplifications, allowing for the detections of AFB1, OTA and ATP with low detection limits. Our assay does not need small molecule-enzyme (or protein) labeling or immobilization of small molecules. This method uses DNA affinity ligands, possessing the merits in preparation, cost, modification, and stability of DNA. The assay is promising for wide applications to detect small molecules with good sensitivity, generality, high throughput, and simplicity.

Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We acknowledged the financial support from National Natural Science Foundation of China (Grant No. 21575153, 21874146, 21435008), the Strategic Priority Research Program of the Chinese Academy of Sciences (XDB14030200). 9



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Supporting Information. Details of reagents, preparation of complementary DNA coated microplate, oligonucleotides sequences, binding buffers for different targets, optimization of experimental conditions; Detection of OTA in the assay using absorbance analysis; Detection of ATP in the assays using absorbance analysis and the chemiluminescence; Tests of selectivity of assays; Detection of targets in complex sample matrix; Comparison of assay performance with reported assays.

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Figure 1. Schematic representation of aptamer structure switch assay coupled with HRP labeling on microplate for small molecule detection. The HRP-labeled aptamer hybridizes with the cDNA immobilized on the microplate surface. HRP catalyzes the substrate into the product, generating absorbance or chemiluminescence signals. The addition of small molecule targets causes the competition between aptamer-target binding and aptamer-cDNA hybridization, leading to signal decrease. Small molecule targets are detected by measuring the signal decrease.

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Blank 10 nM AFB1

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Figure 2. Effect of cDNA with different lengths in aptamer structure switch assay coupled with HRP labeling for AFB1 detection using absorbance analysis. A

3.0 2.7 2.1 1.8 1.5 1.2 0.9

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Figure 4. (A) Aptamer structure switch assay coupled with HRP labeling for OTA detection using the cDNA (C7-OTA) coated microplate and chemiluminescence analysis. The inset showed the signals corresponding to low concentrations of OTA. (B) Responses of aptamer structure switch assay coupled with HRP labeling for detection of ATP in diluted human serum or in the binding buffer by using the cDNA C8-ATP coated microplate and the absorbance analysis.

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Aptamer Structure Switch Coupled with Horseradish Peroxidase

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