Highly Sensitive Detection of Bisphenol A by NanoAptamer Assay with

Subscriber access provided by UB + Fachbibliothek Chemie | (FU-Bibliothekssystem)

Article

Highly sensitive bisphenol A detection by NanoAptamer assay with truncated aptamer Eun-Hee Lee, Hyun Jeong Lim, Sang-Don Lee, and Ahjeong Son ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b02377 • Publication Date (Web): 10 Apr 2017 Downloaded from http://pubs.acs.org on April 11, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 27

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

ACS Applied Materials & Interfaces

Highly sensitive bisphenol A detection by NanoAptamer assay with truncated aptamer Eun-Hee Lee, Hyun Jeong Lim, Sang-Don Lee, Ahjeong Son* Department of Environmental Science and Engineering, Ewha Womans University, Seoul, Republic of Korea

*

Corresponding Author: 52 Ewhayeodae-gil, Seodaemun-gu, Ewha Womans University, Seoul, 120-750, Republic of Korea; E-mail. [email protected]; Phone. +82 (2) 32773339; Fax. +82 (2) 3277-3275.

1

ACS Paragon Plus Environment


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

Abstract We have developed NanoAptamer assay for sensitive quantification of bisphenol A. NanoAptamer assay employs aptamer and complementary signaling DNA, a set of quantum dots and magnetic beads. Signaling DNA - QD655 was tethered to magnetic bead - QD565 via the aptamer. Aptamer affinity with BPA resulted in the release of the signaling DNA - QD655 from the complex and hence corresponding decrease in QD655 fluorescence measurement signal. Three new aptamers (23, 58, and 24-mer) were designed via truncation of the reference aptamer (73-mer). Their respective sensitivity and selectivity of each aptamer for bisphenol A detection via NanoAptamer assay was investigated. One of the truncated aptamers (24-mer) has shown a significantly better performance (limit of detection, LOD 0.17 pg/mL) than the reference 73-mer aptamer (LOD 570 pg/mL). The 24-mer aptamer has also shown the best selectivity of bisphenol A detection over bisphenol A analogs (i.e., bisphenol B, bisphenol C, and diethylstilbestrol). It corresponded to a normalized fluorescence change of 33.7% at environmentally relevant concentration of 1 ng/mL (1 ppb) bisphenol A, while the analogs remained unchanged (2.3 – 3.9%). Keywords: NanoAptamer assay, truncated aptamer, quantum dots, bisphenol A, endocrine disrupting chemicals

Introduction Bisphenol A (2,2-bis (4-hydroxyphenyl) propane) has been widely used in the chemical industry for the synthesis of polycarbonate plastic and epoxy resins. Given the flameretardant, transparent, and thermal-resistant properties,1-3 they are commonly used for consumer products such as nursing bottles, food and beverage packaging, and thermal papers. However, the popular use of bisphenol A has also raised serious concern regarding its implications on food safety and environmental health.2, 4-5 Repeated use of such products results in the leaching of bisphenol A into food and water samples,6-10 at low concentration of ppb levels 1, 11-12 and will eventually find its way into the human body. Unsurprisingly, bisphenol A was found in human blood, tissues, serum, and urine.13-14 As an endocrine disrupting compound15 that exhibits an estrogenic activity, bisphenol A interferes with the hormonal systems by disturbing an estrogen-receptor binding process.16-17 Fortunately, we are gradually phasing out the use of bisphenol A. However the multitudes of used bisphenol A containing products have to be disposed and they often landed in landfills. This means bisphenol A will continue to leach into the environment via runoffs and wastewater discharge and eventually contaminating our water resources and food supplies. It is an understatement that we will need to detect and monitor bisphenol A in the environment. More importantly, it will be beneficial to enable in-situ detection of bisphenol A in relatively inaccessible investigation sites. Conventional chromatographic approach includes liquid chromatography equipped with mass spectrometry (LC/MS) and gas chromatography equipped with mass spectrometry (GC/MS).18-20 Despite their excellent sensitivity and selectivity, the chromatographic methods are ill-suited for deployment in the field due to the current size of the equipment and laborious procedures. The use of aptamer and antibody in conjunction with various transduction methods (e.g., 2


Page 2 of 27

Page 3 of 27

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


fluorescence spectrometry, colorimetry, electrochemistry, and surface plasmon resonance) has been proposed for the in-situ detection of bisphenol A.1, 21-33 Particularly, the aptamer has been considered a promising alternative to antibody in analytics.34-37 Since its discovery in the 1990s, nucleic acid aptamer (e.g., DNA or RNA) has shown relative advantages over antibodies in terms of ease of synthesis and much wider choice of targets. Furthermore, ‘aptamer engineering strategies’ may be employed to re-design the aptamer for a specific target via a particular assay or sensing platform.38-40 This gives rise to unparalleled flexibility and performance.34, 41-42 In this study, we present a NanoAptamer assay based on magnetic beads, quantum dots with a set of new bisphenol A specific aptamers truncated from that previously developed by Jo et al.21 The NanoAptamer assay itself is modified from the NanoGene assay which is known for its in-situ capability43. The NanoAptamer assay consists of magnetic bead-QD565-aptamer bound to signaling DNA-QD655. Upon contact with bisphenol A, the magnetic bead-QD565aptamer captures the bisphenol A and the subsequent aptamer’s conformational change induces the release of signaling DNA-QD655. Hence the concentration of bisphenol A will be directly proportional to the amount of released DNA-QD655 and the corresponding reduction of QD655 fluorescence signal after the magnetic separation. The new truncated aptamers were employed because a shorter aptamer may provide a closer proximity between aptamer and target, hence a better affinity for the target. Prior efforts in performance improvement for aptamer based detection methods have been largely limited to changes in materials, sensor platforms, and analytical procedures.1, 22-32 Yet there has been surprisingly lack of improvement in the design of the aptamer itself.21 Performance of aptamer based detection methods can be largely influenced by the length and structure of the aptamer, which determine the accessibility of target to aptamer. The ssDNA based aptamer forms 3-dimensional secondary structure via the hydrogen bondings between DNA bases. Certain base sequences form the stem-loop structures within aptamer, and they often become the binding sites of targets. However, the exact mechanisms are largely unknown despite of the recent attempts to identify them.44-46 As reviewed by Zhou et al., a full-length aptamer normally comprises three functional regions of binding site, supporting region that facilitates aptamer binding to target, and nonessential nucleotides (neither binding to target nor supporting the recognition of target).47 By cutting off nonessential nucleotides from the aptamer, the binding affinity of the aptamer can be enhanced presumably due to the reduction of steric hindrance.48-49 Specifically, the design and truncation considerations for the new aptamers were first evaluated and implemented. Conformational change of aptamer design was identified by circular dichroism analysis. Sequential formation of aptamer-particle complex was visually verified by Fourier transform-infrared spectrometry and confocal microscope. The potential quenching of QD fluorescence by bisphenol A was also examined. Lastly, the implementation of bisphenol A quantification with regards to sensitivity and selectivity were demonstrated. Quantification of bisphenol A via NanoAptamer assay was implemented in the range of pg/mL (ppt) to ng/mL (ppb) with truncated aptamers. The assay selectivity of the bisphenol A detection was demonstrated by bisphenol A chemical analogs - bisphenol A bisphenol B, bisphenol C, diethylstilbestrol. 3



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

Experimental section Design of aptamers and signaling DNA for bisphenol A detection Single-stranded (ssDNA) aptamers and signaling DNA were designed for NanoAptamer assay. Jo et al. previously developed 63-mer ssDNA aptamer that can bind to bisphenol A21 and the aptamer has been adopted to a number of aptamer based methods.1, 22-26, 28-29, 31-32 With reference to Jo et al.’s aptamer, three truncated aptamers were designed in this study to improve the sensitivity and selectivity of bisphenol A detection. The 30-mer signaling DNA was composed of partially complementary sequences of the 73-mer aptamer (including T10 spacer, will be referred as “the reference aptamer” afterwards) as depicted by blue dotted line in Fig. 1. Three aptamers were truncated from the reference aptamer by considering the potential bisphenol A binding site and the hybridization region between aptamer and signaling DNA. T10 sequence was attached to all aptamers and signaling DNA at 5’ termini to serve as a spacer22 between the oligonucleotides and quantum dots. The truncated aptamers were presented in Table 1 as candidate 1, 2, and 3 and their corresponding base pairs were of 23, 58, and 24-mer, respectively. In Fig. 1, the reference aptamer was first divided into two sections as represented by red dotted box on the left. Both side includes the half of the hybridization region between the reference aptamer and signaling DNA. As a result, two truncated aptamers were generated, and they were designated as candidate 1 and 2 (referred to as 23-mer and 58-mer aptamer below, respectively). The 58-mer aptamer was subsequently shortened by cutting off non-hybridized stem-loop area (represented by red dotted box in the middle section of Fig. 1), resulted in candidate 3 (referred to as 24-mer aptamer below). The sequences of 23-mer and 24-mer aptamers were marginally modified to possess the same secondary structure of the reference aptamer (the modified sequences are depicted by bold face in Table 1). The secondary structures of the newly designed aptamers were predicted using the DNA folding program of Mfold.50 The aptamers and signaling DNA were commercially synthesized with a modification of amine group at 5’ ends (Bioneer Corporation, Daejeon, Korea). Both aptamers and signaling DNA were polyacrylamide gel electrophoresis (PAGE) purified.

Conformational change by circular dichroism spectropolarimetry Circular dichroism (CD) spectropolarimetry is often used to determine the conformation of the aptamer since it is sensitive to structural changes of the DNA.51-53 The result of CD analysis is expressed as a degree of ellipticity (Θ). The CD analysis is able to distinguish the changes within a DNA conformation and isomerization through the changes in ellipticity. CD spectra were recorded at 25ºC using a Jasco J-815 spectropolarimeter (Jasco Inc., Eaton, USA) equipped with a Peltier thermostatic cell holder and nitrogen purging facility. Both aminated and non-aminated aptamers were suspended in 500 µL of 0.02 mole/L Tris-HCl buffer (pH 8.0) containing 0.01% SDS, 20 mmole/L MgCl2, 40 mmole/L KCl, and 100 mmole/L NaCl. The final concentration of aptamers was 2 µmole/L. The aptamer solutions were transferred into a quartz cuvette with optical path length of 10 mm, and its CD spectra were obtained from 350 to 200 nm at a scan rate of 50 nm/min. Note that no differences of CD spectra were observed even though the scan rate increased to 100 nm/min. The spectra were baseline4


Page 4 of 27

Page 5 of 27

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


subtracted with the CD signals from the buffer only. The data were collected from the triplicate scans and all experiments were performed in duplicate. Preparation of MB-QDs particle complexes for bisphenol A detection Magnetic bead (MB) and dual quantum dot nanoparticles (QD565 and QD655) were used to develop NanoAptamer assay suitable for the detection of bisphenol A (Fig. 2). Magnetic bead was used as a carrier of NanoAptamer complex. Two quantum dots were employed to obtain the signals of the aptamer and signaling DNA that were coupled with QD565 and QD655, respectively. Fig. S1 shows scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX) analysis of the NanoAptamer complex (MB-QD565). Note that the magnetic beads are composed of highly cross-linked polystyrene with iron oxide inside the beads, which are ~ 2.5 µm in diameter (Fig. S1a). The quantum dots (QD565 and QD655) are both made of a core nanocrystal (semiconductor material, CdSe), which are shelled with an additional semiconductor layer (ZnS) (Fig. S1b and S1c). The approximate size of QD565 and QD655 are 5 nm and 15 nm, respectively.54 The preparation of particle complexes for NanoAptamer assay is as follows. Aminated magnetic beads (MB, 2 × 109 beads/mL, Dynabead M270, Invitrogen, Carlsbad, USA) were conjugated with carboxyl quantum dot nanoparticles (QD565, 2 µmole/L, Invitrogen, Carlsbad, USA) by forming an amide bond via reaction of ethylcarbodiimide hydrochloride (EDC, Sigma-Aldrich, Saint-Louis, USA) with N-hydroxysuccinimide (NHS, Sigma-Aldrich). Subsequently, 50 pmole of each ssDNA aptamer was covalently attached onto the surface of MB-QD565 particle complex through carboxyl to amine crosslinking between the QD565 and aptamer, which resulted in MB-QD565-aptamer conjugate. One hundred sixty pmole of signaling DNA was separately assembled with carboxyl QD655 nanoparticles (2 µmole/L, Invitrogen) by the mediation of EDC/NHS reaction. The resultant complex (signaling DNAQD655) was mixed with the MB-QD565-aptamer conjugate in 200 µL of 0.02 mole/L Tris-HCl buffer (i.e., the same buffer as used in CD analysis), which allows the DNA hybridization between ssDNA aptamer and signaling DNA. Bisphenol A ( >99%, Daejung, Gyeonggi, Korea) stock solution was prepared in distilled water, and subsequently added into the buffer to achieve a final concentration of 0.0005, 0.001, 0.005, 0.01, 0.05, 0.5, and 5 ng/mL (equivalent to 0.0022, 0.0044, 0.0219, 0.0438, 0.219, 2.19, and 21.9 nmole/L), respectively. Ultrapure distilled water (DNase/RNase/Protease free, Intron Bio-technology, Gyeonggi, Korea) was used as a negative control. The solution was incubated at 25ºC for 16 h under a gentle tilt rotation. The hybrids were washed twice using the hybridization buffer of 0.02 mole/L Tris-HCl to remove unbound or dissociated complex of signaling DNA-QD655 from the MB-QD565-aptamer conjugate. The fluorescence intensity of QD565 and QD655 were measured using a spectrofluorometer (Molecular Devices, SpectraMax M2, Sunnyvale, USA) at 570 and 660 nm of emission wavelengths under the same excitation wavelength of 360 nm, respectively. All experiments were performed in triplicates. The normalized fluorescence (RFU) was determined from the respective fluorescent signals as follow: !"#$$ %&%'(

() =

!"$#$ %&%'(

(1)

Since the aptamer (labeled with QD565) is hybridized with the signaling DNA (labeled with QD655), the fluorescence signals of both QD565 and QD655 are detected in the NanoAptamer 5



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

assay. Once the bisphenol A binds with the aptamer, the following dissociation of signaling DNA occurs. Subsequently, the fluorescence signal of QD655 would decrease while that of QD565 remains the same. The QD565 was used as an internal standard or indicator suitable to confirm whether the NanoAptamer assay complex was successfully assembled or not. Thus, the primary role of the QD565 is to reduce experimental error or to prevent false positive which can occur during the experimental procedure. The normalized fluorescence (QD655 over QD565) will be inversely proportional to the amount of bisphenol A in the NanoAptamer assay. FT-IR analysis The MB and MB-QD565 particles were prepared as potassium bromide (KBr) pellets. The dried particles were analyzed using Fourier transform-infrared spectrometry (FT-IR, Vertex 70, Bruker, Billerica, USA). The FT-IR spectra were obtained with a resolution of 4 cm-1 over the range of 4000 – 400 cm-1. The background of spectra were corrected with the FT-IR signal of the negative control (i.e., KBr pellet only). The amide bonds of MB-QD565 particle complex was identified as compared to MB particles only in order to confirm the covalent bonding between MB and QD565. Confocal microscope analysis Formation of MB-QDs-aptamer complex was visually verified via confocal microscopic images. Three complexes of MB-QD565, MB-QD565-aptamer, and MB-QD565-aptamer hybridized with signaling DNA-QD655 were separately prepared as described above. Note that the aptamer labeled with cyanine dye 5 (Cy5) at the 3’ terminus was employed for the preparation of MB-QD565-aptamer complex. The fluorescent images of QD565, aptamer labeled with Cy5, QD655 were obtained using super resolution confocal microscope (TCS SP8 STED, Leica Microsystems, Wetzlar, Germany) at 570, 680, and 670 nm emission, respectively. Quenching interference of QD fluorescence by bisphenol A Three complexes (MB-QD565, MB-QD565-aptamer, and MB-QD565-aptamer hybridized with signaling DNA-QD655) were tested for the potential QD quenching interference by bisphenol A. Above each three complexes were suspended in 200 µL of 0.02 mole/L Tris-HCl buffer in 1.7 mL of vials in six replicates. Bisphenol A was added to three vials of each three complexes samples to be a final concentration of 5 ng/mL. Ultrapure distilled water was added as a negative control into the other three vials of each complex. All of three complexes samples were incubated at 25ºC for 16 h, and the particles were subsequently washed twice using 200 µL of 0.02 mole/L Tris-HCl buffer. The fluorescence of QD565 and QD655 were measured with the spectrofluorometer. Selectivity of NanoAptamer assay for bisphenol A detection The selectivity of the bisphenol A detection via NanoAptamer assay was validated by analyzing chemical analogs of bisphenol A. As described above, the MB-QD565-aptamer was hybridized with the signaling DNA-QD655 complex in 200 µL of 0.02 mole/L Tris-HCl buffer. Subsequently, bisphenol A and its analogs were added into the solution. Bisphenol B, bisphenol C, diethylstilbestrol ( >98%, all purchased from Tokyo chemical, Tokyo, Japan) 6


Page 6 of 27

Page 7 of 27

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


were selected as analogs of bisphenol A based on their chemical structures. The chemical structure and properties of bisphenol A and analogs were described in Supporting Information (Table S1). Each analog was prepared in distilled water, and added separately to achieve a final concentration of 1 ng/mL (ppb). Bisphenol A and ultrapure distilled water (i.e., no target chemical added) were added as positive and negative controls respectively at the same concentration (1 ppb) for comparison. The fluorescence signals of QD565 and QD655 were measured using spectrofluorometer. All experiments were carried out in triplicates. Detection limit of bisphenol A by NanoAptamer assay The sensitivity of the NanoAptamer assay for bisphenol A detection was demonstrated by determining the limit of detection (LOD). Using the experimental data of Fig. S2, the LOD were estimated in a given analytical procedure described below (2) )* + * * (),+ ) = -./ + 32./

(2)

where XNC and SNC denote the mean and standard deviation of the normalized fluorescence of the negative control, respectively.55-57 Ultrapure distilled water was used as a negative control, and its normalized fluorescence signal was employed to determine LOD value of bisphenol A quantification in the NanoAptamer assay.

Results and Discussion Verification of NanoAptamer assay In order to verify the sequential formation of each component in the NanoAptamer assay, the structures of aptamer-particle complex were analyzed using CD spectropolarimetry, FT-IR spectroscopy, and confocal microscopy. Quenching interference of QD fluorescence by bisphenol A was also examined. With reference to Fig. 3, the CD spectrum of the non-aminated aptamer (closed circles) appeared to be almost identical to that of aminated aptamer (open circles). It indicates that no structural changes occurred during the amine modification of the aptamer. Therefore, it can be concluded that the amine functionalization of the aptamer at 5’ end does not interfere with the aptamer pertaining to the potential formation of secondary structure. As shown in Fig. 4 relative to FT-IR verification, the absorption peaks at 3390 and 1650 /cm were observed in MB particles. They were represented by N-H stretching and N-H bending vibration of the amine groups functionalized on the surface of MB, respectively.58 The peaks at 700 and 1720/cm (depicted by the red circle in Fig. 4b) were only detected in the MBQD565 particle complex (Fig. 4b) as compared to the FT-IR spectrum of MB (Fig. 4a), which represented C=O stretching vibration in the covalent bond. This indicates that the quantum dots (QD565) were successfully coupled with the magnetic beads via the formation of amide bonds. Three complexes (MB-QD565, MB-QD565-aptamer (labeled with Cy5), and MB-QD565aptamer & signaling DNA-QD655) were observed by confocal microscope analysis to validate the conjugation procedure of the NanoAptamer assay. With reference to Fig. 5a, the QD565 7



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

nanoparticles were immobilized on the surface of the magnetic beads which are shown in green color. The attachment of the reference aptamer (Cy5) on the MB-QD565 particle was also optically verified via fluorescent image analysis of Cy5 (shown in red color) which overlapped with QD565 (shown in green color, Fig. 5b). As expected, the fluorescence images of QD565 (represented in green color) and QD655 (represented in red color) were observed in the complex of MB-QD565-aptamer and signaling DNA-QD655 (Fig. 5c). This indicates that the MB-QD565-aptamer complex was well hybridized with the signaling DNA-QD655 conjugate. It was observed that bisphenol A itself did not interfere (i.e., quench) with the fluorescence signal of the quantum dots. As shown in Fig. 6a, the fluorescence intensity of MB-QD565 particles in the presence of bisphenol A was insignificantly different from that in the absence of bisphenol A (p-value = 0.997, based on t-test). Furthermore, the potential conformational change of the aptamer after the binding with bisphenol A also did not seem to influence on the fluorescence signal of MB-QD565-aptamer (p-value = 0.537, Fig. 6b). As for both coexisting MB-QD565-aptamer and signaling DNA-QD655 complex, the fluorescence of QD565 was also not influenced by the presence of bisphenol A (p-value = 0.405, Fig. 6c). Sensitivity of NanoAptamer assay for bisphenol A detection The quantification curve of bisphenol A detection with each truncated aptamer was presented for comparison (Fig. 7). As shown in Fig. 7, all three aptamers have shown the ability to detect bisphenol A. The respective signals (i.e., normalized fluorescence) were proportional to the bisphenol A concentration. However the quantification performance of the NanoAptamer assay for lower concentration of bisphenol A was largely dependent on the size and sequence of the aptamer. It is evident that the normalized fluorescence of the 24-mer aptamer based quantification substantially decreased as the bisphenol A increased (open triangles in Fig. 7). In contrast, the rest did not show such substantial change. With reference to Fig. S2, the use of the 24-mer aptamer gives a regression of y = -0.23 log10 x + 0.74 where the normalized fluorescence (S) is plotted in y-axis and the bisphenol A concentration (C) in x-axis. The range of bisphenol A quantification using the 24-mer aptamer was ~4 orders of magnitude (100 – 104 pg/mL) as shown in Fig. S2d. The regression for 23-mer, and 58-mer aptamers of y = -0.023 log10 x + 0.72, y = -0.019 log10 x + 0.65, and y = -0.020 log10 x + 0.57, respectively (Fig. S2). The slope (∆S/∆C) of the regression was in a range of -0.019 – -0.23 and the y-intercepts were from 0.57 to 0.72 for the four sensors. It should be noted that the 24-mer aptamer (candidate 3) showed the largest slope (-0.23) which is approximately tenfold greater than those of other aptamers. Since the sensor’s signal is proportional to the dissociation of the signaling DNA-QD655 from the binding of aptamer to bisphenol A, a larger slope would imply a better performance of the assay. In order to validate that the truncated aptamers have affinity to bisphenol A, a CD analysis was performed to elucidate their respective conformational changes upon binding with bisphenol A. As shown in Fig. S3, all truncated aptamers have shown changes in their respective CD spectra in accordance to the amount of bisphenol A added. In particular, the 24-mer aptamer (Fig. S3c) showed significant change in its CD spectrum as compared to the other two aptamers. The 24-mer aptamer showed a change of ~20.6% at 278 nm that corresponded to an additional 10 ng/mL bisphenol A (Fig. S3c), while the other truncated aptamers (23-mer and 58-mer aptamers) showed changes of ~9.8% (Fig. S3a) and ~7.8% (Fig. S3b), respectively. As shown in Fig. 7, 8


Page 8 of 27

Page 9 of 27

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


the sensitivity result of the NanoAptamer assay has also shown that the 24-mer aptamer has the strongest affinity for bisphenol A as compared to the other truncated aptamers. In other words, the CD analysis results are consistent with that of the NanoAptamer assay. Hence, this consistency of results and the conformal changes observed in the CD analysis validated the affinity of the truncated aptamers for bisphenol A. Using the experimental data in Figs. 7 and S2, the LOD was determined with equation (2) described earlier.55-57 With reference to Table 2, the NanoAptamer assay with the reference aptamer showed the LOD of 570 pg/mL, while the assay with the 23-mer and 58-mer aptamers (candidate 1 and 2) had the LOD of 920 and 1900 pg/mL bisphenol A, respectively. It is noted that the NanoAptamer assay with 24-mer aptamer (candidate 3) had significantly better performance (LOD of 0.17 pg/mL). This indicates that the 24-mer aptamer may be superior to the other aptamers (reference, 23-mer, and 58-mer aptamers) for bisphenol A detection via NanoAptamer assay. In other words, the 24-mer aptamer may have retained the region that enables the effective capturing of bisphenol A. As indicated in Table 2, the LOD level of 0.17 pg/mL by the NanoAptamer assay with the 24mer aptamer is lower than the previous studies so far. Kuang et al. developed a plasmonic chirality-based aptasensor for bisphenol A detection and obtained LOD of 8 pg/mL.22 Xue et al. presented an electrochemical aptasensor to determine bisphenol A in drinking water and it was capable of detecting bisphenol A as low as 0.284 pg/mL.28 Kang et al. described an anodized aluminum oxide-based capacitive sensor, and demonstrated its bisphenol A detection as low as 23 pg/mL.31 In comparison to above studies, it can be concluded that the 24-mer aptamer based NanoAptamer assay achieved the most sensitive detection among similar approaches. It is important to note that the other aptamer based methods utilize the reference Jo’s aptamer,21 while the assay in this study uses the newly truncated aptamer. Moreover, the LOD of 0.17 pg/mL bisphenol A was less than desirable environmentally relevant levels (i.e., 50 – 1614 pg/mL).59-60 It indicates that the developed NanoAptamer assay with the truncated aptamer may satisfy the analytical needs for environmental samples pertaining to bisphenol A detection. Based on the result shown in Fig. 7, the detection capability of the NanoAptamer assay with 24-mer aptamer was significantly better than those of the reference and 58-mer aptamers. The plausible explanation is that oligo structure and length of the aptamers may influence the sensor’s capability by affecting the accessibility of bisphenol A to aptamer. As for the stemloop structure, the reference and 58-mer aptamers contain the same stem-loop area as the 24mer aptamer retains (depicted by the red dotted box in the middle section of Fig.1). At the same time, the reference and 58-mer aptamers possess other extra stem-loop regions as compared to the 24-mer aptamer (depicted by the blue dotted box in the middle section of Fig. 1). If the common step-loop structure (red dotted box) of all three aptamer is the main bisphenol A binding site, the extra step-loop structures (blue dotted box) may hinder the accessibility of bisphenol A to the aptamer by decreasing a proximity between bisphenol A and aptamer. In other words, the 24-mer aptamer with the better performance may contain the core sequence region that participates in bisphenol A binding, and the unnecessary sequence region has been removed. Selectivity of NanoAptamer assay for bisphenol A detection 9



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

The selectivity of the NanoAptamer assay with various aptamers for bisphenol A detection was demonstrated using its chemical analogs (bisphenol B, bisphenol C, and diethylstilbestrol). The chemical structures of bisphenol A analogs were described in Table S1. As expected, the four aptamers have shown different selectivity toward the bisphenol A detection (Figs. 8a – 8d). The NanoAptamer assay with 24-mer aptamer was able to detect bisphenol A with the highest selectivity (circle in Fig. 8d). The normalized fluorescence decreased only with bisphenol A which corresponded to a decrease of 33.7% from the fluorescence signal of the negative control (i.e., no target chemical is added). This change was statistically significant as compared to the negative control (p-value = 0.046). On the other hand, the normalized fluorescence that corresponded to bisphenol B, bisphenol C, and diethylstilbestrol remained unchanged (2.3 – 3.9% decrease, p-values = 0.789 (bisphenol B), 0.698 (bisphenol C), and 0.331 (diethylstilbestrol), respectively, based on t-test) as compared to the negative control. The reference aptamer in Fig. 8a also appeared to be selective in detecting bisphenol A. The signal of bisphenol A detection decreased 18.7% of negative control. This means the selectivity of the reference aptamer is slightly worse than the 24-mer aptamer in Fig. 8d (18.7% vs. 33.7%). The difference is probably attributed to the different binding affinity of the aptamer to bisphenol A. On the other hand, the 23-mer (Fig. 8b) and 58-mer (Fig. 8c) aptamers have shown much worse selectivity for the detection of bisphenol A as compared to 24-mer aptamer. The 23-mer aptamer has shown a decreased signal toward bisphenol C (depicted by the arrow in Fig. 8b). The decreased signals of 11.3% and 12.0% correspond to bisphenol A and bisphenol C, respectively. The similar value of decreased signal indicates that 23-mer aptamer may not be able to differentiate between bisphenol A and bisphenol C. In other words, the 23-mer aptamer showed the poor selectivity of bisphenol A detection toward bisphenol C. The 58-mer aptamer has also shown the similar pattern of the poor selectively toward diethylstilbestrol. Diethylstilbestrol detection by 58-mer aptamer showed a decrease of 8.3% from the negative control (depicted by the arrow in Fig. 8c), while it showed a decrease of 16.0% for bisphenol A. This indicates that the NanoAptamer assay with 24-mer truncated aptamer has a considerably high selectivity for bisphenol A detection as compared to the other aptamers. Note that the 24-mer aptamer includes T10 sequences as a spacer between QD565 and aptamer. If the T10 sequences are excluded, only 14 nucleotides remain as an aptamer region that can selectively bind to bisphenol A. This is a much shorter oligo length as compared to the reference aptamer (63-mer).21 The 24-mer aptamer may retain the minimal sequence of ligand binding domain which is required for the binding to bisphenol A.

Conclusions We have demonstrated NanoAptamer assay capable of highly sensitive detection of bisphenol A with truncated aptamer. Among new three truncated aptamers, the 24-mer aptamer has shown a significantly better sensitivity (LOD = 0.17 pg/mL) compared to the reference aptamer (LOD = 570 pg/mL). This is much lower than environmentally relevant bisphenol A 10


Page 10 of 27

Page 11 of 27

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


concentration of ppb level. The range of quantification was about 4 orders of magnitude (100 – 104 pg/mL). The 24-mer aptamer has also shown the best selectivity of bisphenol A detection over bisphenol A analogs (i.e., bisphenol B, bisphenol C, and diethylstilbestrol). It corresponded to a normalized fluorescence change of 33.7% at 1 ppb bisphenol A, while the analogs remained unchanged (2.3 – 3.9%). To the best our knowledge, the 24-mer aptamer (14-mer without T10 spacer) is the shortest aptamer with the highest affinity and selectivity for the bisphenol A detection thus far. The information gained in this study suggests that the NanoAptamer assay can be a valuable laboratory method that can be transplanted into a portable system for bisphenol A detection. And the newly truncated aptamer can be extended to other detection platforms for ultrasensitive detection of bisphenol A.

Associated content Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Chemical properties of bisphenol A and its analogs, FE-SEM and EDX analysis of NanoAptamer assay, sensitivity of NanoAptamer assay with different aptamers for the detection of bisphenol A, CD spectral changes of truncated aptamers due to binding with bisphenol A (PDF). Notes The authors declare no competing financial interest.

Acknowledgements This project was made possible by funding provided by Ministry of Science, ICT, and Future Planning in Korea (2015M3C8A6A06012735).

References (1) Yildirim, N.; Long, F.; He, M.; Shi, H. C.; Gu, A. Z. A Portable Optic Fiber Aptasensor for Sensitive, Specific and Rapid Detection of Bisphenol-A in Water Samples. Environ. Sci.: Processes. Impacts. 2014, 16 (6), 1379-1386. (2) Liao, C. Y.; Kannan, K. Widespread Occurrence of Bisphenol A in Paper and Paper Products: Implications for Human Exposure. Environ. Sci. Technol. 2011, 45 (21), 9372-9379. (3) Bailin, P. D.; Byrne, M.; Lewis, S.; Liroff, R. Public Awareness Drives Market for Safer Alternatives: Bisphenol A Market Analysis Report. http://www.iehn.org/publications.reports.bpa.php 2008. (4) Ragavan, K. V.; Rastogi, N. K.; Thakur, M. S. Sensors and Biosensors for Analysis of Bisphenol-A. Trac-Trend. Anal. Chem. 2013, 52, 248-260. (5) Alonso-Magdalena, P.; Ropero, A. B.; Soriano, S.; Quesada, I.; Nadal, A. Bisphenol11



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

A: A New Diabetogenic Factor? Horm.-Int. J. Endocrino. 2010, 9 (2), 118-126. (6) Ehlert, K. A.; Beumer, C. W. E.; Groot, M. C. E. Migration of Bisphenol A into Water from Polycarbonate Baby Bottles During Microwave Heating. Food. Addit. Contam. A. 2008, 25 (7), 904-910. (7) Cao, X. L.; Corriveau, J. Migration of Bisphenol A from Polycarbonate Baby and Water Bottles into Water under Severe Conditions. J. Agric. Food. Chem. 2008, 56 (15), 6378-6381. (8) Lim, D. S.; Kwack, S. J.; Kim, K. B.; Kim, H. S.; Lee, B. M. Potential Risk of Bisphenol A Migration from Polycarbonate Containers after Heating, Boiling, and Microwaving. J. Toxicol. Environ. Health, Part A 2009, 72 (21-22), 1285-1291. Goodson, A.; Robin, H.; Summerfield, W.; Cooper, I. Migration of Bisphenol A from (9) Can Coatings - Effects of Damage, Storage Conditions and Heating. Food Addit. Contam. 2004, 21 (10), 1015-1026. (10) Schonfelder, G.; Wittfoht, W.; Hopp, H.; Talsness, C. E.; Paul, M.; Chahoud, I. Parent Bisphenol A Accumulation in the Human Maternal-Fetal-Placental Unit. Environ. Health Perspect. 2002, 110 (11), A703-A707. (11) Dekant, W.; Voelkel, W. Human Exposure to Bisphenol A by Biomonitoring: Methods, Results and Assessment of Environmental Exposures. Toxicol. Appl. Pharmacol. 2008, 228 (1), 114-134. (12) Volkel, W.; Kiranoglu, M.; Fromme, H. Determination of Free and Total Bisphenol A in Human Urine to Assess Daily Uptake as a Basis for a Valid Risk Assessment. Toxicol. Lett. 2008, 179 (3), 155-162. Vandenberg, L. N.; Hauser, R.; Marcus, M.; Olea, N.; Welshons, W. V. Human (13) Exposure to Bisphenol A (BPA). Reprod. Toxicol. 2007, 24 (2), 139-177. (14) Welshons, W. V.; Nagel, S. C.; vom Saal, F. S. Large Effects from Small Exposures. Iii. Endocrine Mechanisms Mediating Effects of Bisphenol A at Levels of Human Exposure. Endocrinology 2006, 147 (6), S56-S69. (15) Rubin, B. S. Bisphenol A: An Endocrine Disruptor with Widespread Exposure and Multiple Effects. J. Steroid Biochem. Mol. Biol. 2011, 127 (1-2), 27-34. (16) Diamanti-Kandarakis, E.; Bourguignon, J. P.; Giudice, L. C.; Hauser, R.; Prins, G. S.; Soto, A. M.; Zoeller, R. T.; Gore, A. C. Endocrine-Disrupting Chemicals: An Endocrine Society Scientific Statement. Endocr. Rev. 2009, 30 (4), 293-342. Swedenborg, E.; Ruegg, J.; Makela, S.; Pongratz, I. Endocrine Disruptive Chemicals: (17) Mechanisms of Action and Involvement in Metabolic Disorders. J. Mol. Endocrinol. 2009, 43 (1-2), 1-10. (18) Stuart, J. D.; Capulong, C. P.; Launer, K. D.; Pan, X. J. Analyses of Phenolic Endocrine Disrupting Chemicals in Marine Samples by Both Gas and Liquid Chromatography-Mass Spectrometry. J. Chromatogr. A 2005, 1079 (1-2), 136-145. (19) Ballesteros-Gomez, A.; Rubio, S.; Perez-Bendito, D. Analytical Methods for the Determination of Bisphenol A in Food. J. Chromatogr. A 2009, 1216 (3), 449-469. (20) Rezaee, M.; Yamini, Y.; Shariati, S.; Esrafili, A.; Shamsipur, M. Dispersive LiquidLiquid Microextraction Combined with High-Performance Liquid Chromatography-UV Detection as a Very Simple, Rapid and Sensitive Method for the Determination of Bisphenol A in Water Samples. J. Chromatogr. A 2009, 1216 (9), 1511-1514. (21) Jo, M.; Ahn, J. Y.; Lee, J.; Lee, S.; Hong, S. W.; Yoo, J. W.; Kang, J.; Dua, P.; Lee, D. K.; Hong, S.; Kim, S. Development of Single-Stranded DNA Aptamers for Specific 12


Page 12 of 27

Page 13 of 27

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


Bisphenol A Detection. Oligonucleotides 2011, 21 (2), 85-91. (22) Kuang, H.; Yin, H.; Liu, L.; Xu, L.; Ma, W.; Xu, C. Asymmetric Plasmonic Aptasensor for Sensitive Detection of Bisphenol A. ACS Appl. Mater. Interfaces 2014, 6 (1), 364-369. (23) Yao, D. M.; Wen, G. Q.; Jiang, Z. L. A Highly Sensitive and Selective Resonance Rayleigh Scattering Method for Bisphenol A Detection Based on the Aptamer-Nanogold Catalysis of the Haucl4-Vitamin C Particle Reaction. RSC Adv. 2013, 3 (32), 13353-13356. (24) Marks, H. L.; Pishko, M. V.; Jackson, G. W.; Cote, G. L. Rational Design of a Bisphenol A Aptamer Selective Surface-Enhanced Raman Scattering Nanoprobe. Anal. Chem. 2014, 86 (23), 11614-11619. Zhang, Y. Y.; Cao, T. C.; Huang, X. F.; Liu, M. C.; Shi, H. J.; Zhao, G. H. A Visible(25) Light Driven Photoelectrochemical Aptasensor for Endocrine Disrupting Chemicals Bisphenol A with High Sensitivity and Specificity. Electroanal. 2013, 25 (7), 1787-1795. (26) Zhu, Y. Y.; Cai, Y. L.; Xu, L. G.; Zheng, L. X.; Wang, L. M.; Qi, B.; Xu, C. L. Building an Aptamer/Graphene Oxide Fret Biosensor for One-Step Detection of Bisphenol A. ACS Appl. Mater. Interfaces 2015, 7 (14), 7492-7496. (27) Zhou, L.; Wang, J. P.; Li, D. J.; Li, Y. B. An Electrochemical Aptasensor Based on Gold Nanoparticles Dotted Graphene Modified Glassy Carbon Electrode for Label-Free Detection of Bisphenol A in Milk Samples. Food Chem. 2014, 162, 34-40. (28) Xue, F.; Wu, J. J.; Chu, H. Q.; Mei, Z. L.; Ye, Y. K.; Liu, J.; Zhang, R.; Peng, C. F.; Zheng, L.; Chen, W. Electrochemical Aptasensor for the Determination of Bisphenol A in Drinking Water. Microchim. Acta 2013, 180 (1-2), 109-115. Lee, J.; Jo, M.; Kim, T. H.; Ahn, J. Y.; Lee, D. K.; Kim, S.; Hong, S. Aptamer (29) Sandwich-Based Carbon Nanotube Sensors for Single-Carbon-Atomic-Resolution Detection of Non-Polar Small Molecular Species. Lab Chip 2011, 11 (1), 52-56. (30) Ragavan, K. V.; Selvakumar, L. S.; Thakur, M. S. Functionalized Aptamers as NanoBioprobes for Ultrasensitive Detection of Bisphenol-A. Chem. Commun. 2013, 49 (53), 59605962. (31) Kang, B.; Kim, J. H.; Kim, S.; Yoo, K. H. Aptamer-Modified Anodized Aluminum Oxide-Based Capacitive Sensor for the Detection of Bisphenol A. Appl. Phys. Lett. 2011, 98 (7), 073703. (32) Mei, Z. L.; Chu, H. Q.; Chen, W.; Xue, F.; Liu, J.; Xu, H. N.; Zhang, R.; Zheng, L. Ultrasensitive One-Step Rapid Visual Detection of Bisphenol A in Water Samples by LabelFree Aptasensor. Biosens. Bioelectron. 2013, 39 (1), 26-30. (33) Mei, Z. L.; Qu, W.; Deng, Y.; Chu, H. Q.; Cao, J. X.; Xue, F.; Zheng, L.; El-Nezamic, H. S.; Wu, Y. C.; Chen, W. One-Step Signal Amplified Lateral Flow Strip Biosensor for Ultrasensitive and on-Site Detection of Bisphenol A (BPA) in Aqueous Samples. Biosens. Bioelectron. 2013, 49, 457-461. (34) Ilgu, M.; Nilsen-Hamilton, M. Aptamers in Analytics. Analyst 2016, 141 (5), 15511568. (35) Song, K. M.; Lee, S.; Ban, C. Aptamers and Their Biological Applications. Sensors 2012, 12 (1), 612-631. (36) Ruigrok, V. J. B.; Levisson, M.; Eppink, M. H. M.; Smidt, H.; van der Oost, J. Alternative Affinity Tools: More Attractive Than Antibodies? Biochem. J. 2011, 436, 1-13. (37) Proske, D.; Blank, M.; Buhmann, R.; Resch, A. Aptamers - Basic Research, Drug Development, and Clinical Applications. Appl. Microbiol. Biotechnol. 2005, 69 (4), 367-374. 13



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

(38) Zhang, J.; Wang, L.; Pan, D.; Song, S.; Boey, F. Y.; Zhang, H.; Fan, C. Visual Cocaine Detection with Gold Nanoparticles and Rationally Engineered Aptamer Structures. Small 2008, 4 (8), 1196-1200. (39) Wen, Y.; Pei, H.; Wan, Y.; Su, Y.; Huang, Q.; Song, S.; Fan, C. DNA NanostructureDecorated Surfaces for Enhanced Aptamer-Target Binding and Electrochemical Cocaine Sensors. Anal. Chem. 2011, 83 (19), 7418-7423. (40) Zuo, X.; Xiao, Y.; Plaxco, K. W. High Specificity, Electrochemical Sandwich Assays Based on Single Aptamer Sequences and Suitable for the Direct Detection of Small-Molecule Targets in Blood and Other Complex Matrices. J. Am. Chem. Soc. 2009, 131 (20), 6944-6945. (41) Liu, J. C.; Bai, W. H.; Niu, S. C.; Zhu, C.; Yang, S. M.; Chen, A. L. Highly Sensitive Colorimetric Detection of 17 Beta-Estradiol Using Split DNA Aptamers Immobilized on Unmodified Gold Nanoparticles. Sci. Rep. 2014, 4, 7571. (42) Kwon, Y. S.; Raston, N. H. A.; Gu, M. B. An Ultra-Sensitive Colorimetric Detection of Tetracyclines Using the Shortest Aptamer with Highly Enhanced Affinity. Chem. Commun. 2014, 50 (1), 40-42. Mitchell, K. A.; Chua, B.; Son, A. Development of First Generation In-Situ Pathogen (43) Detection System (Gen1-IPDS) Based on Nanogene Assay for near Real Time E. coli O157:H7 Detection. Biosens. Bioelectron. 2014, 54, 229-236. (44) Li, L. L.; Wang, Q. Q.; Zhang, Y.; Niu, Y. Z.; Yao, X. J.; Liu, H. X. The Molecular Mechanism of Bisphenol A (BPA) as an Endocrine Disruptor by Interacting with Nuclear Receptors: Insights from Molecular Dynamics (MD) Simulations. PLoS One 2015, 10 (3), e0120330. Yunn, N. O.; Koh, A.; Han, S.; Lim, J. H.; Park, S.; Lee, J.; Kim, E.; Jang, S. K.; (45) Berggren, P. O.; Ryu, S. H. Agonistic Aptamer to the Insulin Receptor Leads to Biased Signaling and Functional Selectivity through Allosteric Modulation. Nucleic Acids Res. 2015, 43 (16), 7688-7701. (46) Durand, G.; Dausse, E.; Goux, E.; Fiore, E.; Peyrin, E.; Ravelet, C.; Toulme, J. J. A Combinatorial Approach to the Repertoire of Rna Kissing Motifs; Towards Multiplex Detection by Switching Hairpin Aptamers. Nucleic Acids Res. 2016, 44 (9), 4450-4459. (47) Zhou, J.; Battig, M. R.; Wang, Y. Aptamer-Based Molecular Recognition for Biosensor Development. Anal. Bioanal. Chem. 2010, 398 (6), 2471-2480. Zhou, J.; Soontornworajit, B.; Snipes, M. P.; Wang, Y. Structural Prediction and (48) Binding Analysis of Hybridized Aptamers. J. Mol. Recognit. 2011, 24 (1), 119-126. (49) White, R. R.; Shan, S.; Rusconi, C. P.; Shetty, G.; Dewhirst, M. W.; Kontos, C. D.; Sullenger, B. A. Inhibition of Rat Corneal Angiogenesis by a Nuclease-Resistant RNA Aptamer Specific for Angiopoietin-2. Proc. Natl. Acad. Sci. U. S. A. 2003, 100 (9), 50285033. Zuker, M. Mfold Web Server for Nucleic Acid Folding and Hybridization Prediction. (50) Nucleic Acids Res. 2003, 31 (13), 3406-3415. (51) Kypr, J.; Kejnovska, I.; Renciuk, D.; Vorlickova, M. Circular Dichroism and Conformational Polymorphism of DNA. Nucleic Acids Res. 2009, 37 (6), 1713-1725. (52) Vorlickova, M.; Kejnovska, I.; Bednarova, K.; Renciuk, D.; Kypr, J. Circular Dichroism Spectroscopy of DNA: From Duplexes to Quadruplexes. Chirality 2012, 24 (9), 691-698. (53) Chang, Y. M.; Chen, C. K. M.; Hou, M. H. Conformational Changes in DNA Upon Ligand Binding Monitored by Circular Dichroism. Int. J. Mol. Sci. 2012, 13 (3), 3394-3413. 14


Page 14 of 27

Page 15 of 27

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


(54) Whaley, P. Qdot® Conjugates: Sensitive, Multicolor, Stable Fluorescence, Invitrogen. (https://www.thermofisher.com). (55) Armbruster, D. A.; Pry, T. Limit of Blank, Limit of Detection and Limit of Quantitation. Clin. Biochem. Rev. 2008, 29 Suppl 1, S49-52. (56) Analytical Methods, C. Recommendations for the Definition, Estimation and Use of the Detection Limit. Analyst 1987, 112 (2), 199-204. (57) Shrivastava, A.; Gupta, V. B. Methods for the Determination of Limit of Detection and Limit of Quantitation of the Analytical Methods. Chron. Young Sci. 2011, 2 (1), 21. (58) Pavia, D. L.; Lampman, G. M.; Kriz, G. S.; Vyvyan, J. A., Introduction to Spectroscopy. Cengage Learning: 2008. (59) Guerra, P.; Kim, M.; Teslic, S.; Alaee, M.; Smyth, S. A. Bisphenol-A Removal in Various Wastewater Treatment Processes: Operational Conditions, Mass Balance, and Optimization. J. Environ. Manage. 2015, 152, 192-200. (60) Kassotis, C. D.; Alvarez, D. A.; Taylor, J. A.; vom Saal, F. S.; Nagel, S. C.; Tillitt, D. E. Characterization of Missouri Surface Waters Near Point Sources of Pollution Reveals Potential Novel Atmospheric Route of Exposure for Bisphenol A and Wastewater Hormonal Activity Pattern. Sci. Total Environ. 2015, 524-525, 384-393.

15



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

Figure legends Figure 1. Proposed secondary structures of reference aptamer and truncated aptamers for bisphenol A detection. Figure 2. Schematics of NanoAptamer assay suitable for bisphenol A detection. Conceptual design and procedure diagram were described for the bisphenol A detection via NanoAptamer assay. Figure 3. Comparison of CD spectra of aminated- with non-aminated aptamer. The reference aptamer was used for this analysis. Figure 4. FT-IR spectra of (a) MB and (b) MB-QD565 particle complex. Figure 5. Confocal microscope images of each preparation step of NanoAptamer assay. (a) MB-QD565, (b) MB-QD565-aptamer (labeled with Cy5) (c) MB-QD565-aptamer & signaling DNA-QD655 conjugates. Figure 6. Effects of bisphenol A on the fluorescence of quantum dot nanoparticles. (a) MBQD565 (b) MB-QD565-aptamer, and (c) MB-QD565-aptamer & signaling DNA-QD655 conjugates. Error bars indicate the standard deviations of triplicate samples, and the same description applies to Figures 7 and 8. Figure 7. Sensitivity comparison of NanoAptamer assay for the detection of bisphenol A. The four aptamers (reference, 23-, 58-, and 24-mer aptamers) were employed for the development of NanoAptamer assay and the comparison of their sensitivity. Note that ultrapure distilled water was used as a negative control and the results are depicted by blue markers at 0 ng/mL of bisphenol A in x-axis. Figure 8. Comparison of selectivity of the developed NanoAptamer assay with (a) reference aptamer, (b) 23-mer aptamer, (c) 58-mer aptamer, and (d) 24-mer aptamer. Bisphenol B, bisphenol C, and diethylstilbestrol are the chemical analogs of bisphenol A.

16


Page 16 of 27

Page 17 of 27

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


Table 1. Sequences of ssDNA aptamer and signaling DNA oligonucleotides used in this study. Oligonucleotides

Reference aptamer

Truncated aptamers

Signaling DNA a

Length (mer) 73 (63)a

Sequence (5’ to 3’)

Reference

NH2-C6-(T)10-CCGGTGGGTG GTCAGGTGGG ATAGCGTTCC GCGTATGGCC

1, 2

CAGCGCATCA CGGGTTCGCA CCA

Candidate 1

23 (13)

NH2-C6-(T)10-CCGGTGGGTG GAA

Candidate 2

58 (48)

Candidate 3

24 (14)

NH2-C6-(T)10-GGATAGCGGG TTCC

30

NH2-C6-(T)10-TATCCCACCT GACCACCCAC

NH2-C6-(T)10-GGTGGGATAG

CGTTCCGCGT

ATGGCCCAGC

GCATCACGGG TTCGCACC

The length of aptamer excluding a spacer of T10 sequences.

The modified sequences of truncated aptamers as compared to the reference aptamer were depicted by bold face. The underlined sequences refer to the complementary regions between the aptamers and signaling DNA.

17


This study


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

Page 18 of 27

Table 2. Summary of detection limit of bisphenol A quantification by aptamer based methods. Methods Portable optic fiber aptasensor using fluorescent labeled aptamer Plasmonic chiral aptasensor Resonance Rayleigh scattering Surface-enhanced Raman scattering nanoprobes Photoelectrochemical aptasensor Aptamer/graphene oxide FRET biosensor GNPs and graphene based electrochemical aptasensor Electrochemical aptasensor Sandwich-based carbon nanotube sensor Anodized aluminum oxide-based capacitive sensor GNPs aggregation based colorimetric aptasensor Magnetic beads and quantum dots based NanoAptamer assay a

Aptamer size (mer) 63 73 (63)a 108 60 108 63 63 63 108 108 63 73 (63) a 23 (13) a 58 (48) a 24 (14) a

The length of aptamer excluding a spacer of T10 sequences.

18


Limit of detection (pg/mL) 450 8 83 750 4 50 1200 0.284 0.23 23 100 570 920 1900 0.17

Reference 1 22 23 24 25 26 27 28 29 31 32

This study

Page 19 of 27

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


Figure 1

19



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

Figure 2 20


Page 20 of 27

Page 21 of 27

12 10

θ , mdeg) Ellipticity (θ

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


Non-aminated aptamer Aminated aptamer

8 6 4 2 0 -2 -4 -6 200

225

250

275

300

Wavelength (nm) Figure 3

21


325

350


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

Figure 4

22


Page 22 of 27

Page 23 of 27

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


Figure 5

23



Fluorescence (QD565, RFU)

250

(a) MB-QD565 200

150

100

50

0 Without bisphenol A

With bisphenol A


250

(b) MB-QD565-aptamer 200

150

100

50

0 Without bisphenol A

With bisphenol A

350


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

300

(c) MB-QD 565-aptamer & signaling DNA-QD 655

250 200 150 100 50 0 Without bisphenol A

With bisphenol A

Figure 6 24


Page 24 of 27

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


Normalized fluorescence (QD655/QD565)

Page 25 of 27

2.4 2.2

Reference aptamer 23-mer aptamer 58-mer aptamer 24-mer aptamer

2.0 1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 100-4

10-3

10-2

10-1

100

Bisphenol A concentration (ng/mL) Figure 7

25


101

12

(a)

NanoAptamer assay using reference aptamer

10

8

6

4

2

0


e co ativ N eg

l A C B trol ntro n ol n ol no l bes p he p he p he lstil B is B is B is thy ie D

8

(b)

NanoAptamer assay using 23-mer aptamer

6

4

2

e co ativ Neg

l l A C B stro ntro nol nol no l ilbe p he ph e phe ylst Bis Bis Bis h t Die


0


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



8

(c)


6

4

2

0

Ne

c ive gat

l A B C rol stro no l n ol n ol o nt ilbe ph e p he phe ylst Bis B is Bis h t D ie

10

(d)


8

6

4

2

0 ativ N eg

l l A B C stro ntro no l nol n ol ilbe e co phe phe phe ylst B is Bis B is h t D ie

Figure 8

26


Page 26 of 27

Page 27 of 27

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


Table of Contents

27


Highly Sensitive Detection of Bisphenol A by NanoAptamer Assay with

Recommend Documents