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Target-recycling-amplified colorimetric detection of pollen allergen using non-cross-linking aggregation of DNA-modified gold nanoparticles Chia-Chen Chang, Guoqing Wang, Tohru Takarada, and Mizuo Maeda ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b01156 • Publication Date (Web): 10 Jan 2019 Downloaded from http://pubs.acs.org on January 11, 2019
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Target-recycling-amplified colorimetric detection of pollen allergen using non-cross-linking aggregation of DNA-modified gold nanoparticles Chia-Chen Chang,*,†,‡ Guoqing Wang,†,§ Tohru Takarada,*,† and Mizuo Maeda† †Bioengineering
Laboratory, RIKEN Cluster for Pioneering Research, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
‡Biomedical
Technology and Device Research Laboratories, Industrial Technology Research Institute, Chutung, Hsinchu 31057, Taiwan §College
of Food Science and Engineering, Ocean University of China, 5 Yushan Road, Qingdao 266003, China
KEYWORDS Allergen, Aptamer, Cry j 2, DNA, Gold nanoparticle, Surface plasmon resonance
ABSTRACT: Increasing prevalence of pollen allergies has raised concerns about human health. Development of a facile and precise method to detect pollen allergens would thus be of significance for environmental assessments and medical diagnoses. Here we report a sensitive colorimetric method to detect the Japanese cedar pollen allergen, Cry j 2. The method consists of two steps: a signal amplification based on the catalytic DNA hairpin self-assembly, followed by a signal transduction using the salt-induced non-cross-linking aggregation of gold nanoparticles densely modified with short DNA. The assay exhibits a detection limit of 0.2 ng/mL, which is 130-fold greater than that of the previously reported one. Moreover, the assay enables the detection of Cry j 2 spiked in soil solutions by avoiding any interference from the contaminants. The signal amplification system includes an anti-Cry j 2 DNA aptamer, which accounts for the absence of false responses to five non-target allergen proteins. The present method could be of general applicability to various proteins by using appropriate aptamers.
Over the last several decades, the lifetime prevalence of pollinosis has gradually increases worldwide.1 Japanese cedar (Cryptomeria japonica) has been considered a major source of allergenic pollen in Japan. The estimated prevalence rate of Japanese cedar pollinosis in Tokyo was 28.2% in 2006.2 These facts underscore the necessity of monitoring the exposure level of Japanese cedar pollen, particularly for on-site environmental assessments. Although a colorimetric enzyme-linked immunosorbent assay has been widely used to detect allergens with a high degree of sensitivity and specificity,3 a number of issues including the long time required for measurement, the relative complexity of the procedure, and the limited storage of reagents should be addressed for on-site environmental monitoring. Most of these issues arise from the use of proteins (antibodies and enzymes) as the functional elements of assays. Accordingly, DNA/RNA aptamers have often been employed, instead of antibodies, as a target recognition unit of biosensors.4-6 For example, a DNA aptamer-based blotting assay to detect Japanese cedar pollen allergen, Cry j 2 (polymethylgalacturonase, 37 kDa, pI 9–10), was recently reported by Ogihara et al.7 Their assay exhibited great simplicity and stability, but its sensitivity must be further improved before practical use.
In the present study, we used a DNA aptamer as a recognition unit to achieve the short time for the measurement and the simplicity of the procedure. To further improve the sensitivity, we sought to construct a colorimetric assay for Cry j 2 by adopting a signal amplification strategy based on catalytic DNA hairpin selfassembly. This amplification strategy has been attracting considerable attention due to its robustness and low cost.810 The signal amplification process is designed to contain an anti-Cry j 2 DNA aptamer for a target recognition unit. To draw visual responses to sample solutions, we combine the signal amplification with colloidal aggregation of gold nanoparticles (AuNPs). In general, AuNP-based assays can transduce a target-induced molecular event which realizes interparticle plasmon coupling, and ultimately a solution color change from red to purple.11-13 The present study utilizes salt-induced aggregation of AuNPs densely modified with double-stranded DNA (dsDNA–AuNPs), which was identified by Sato et al.14 Although single-stranded (ss) DNA–AuNPs can disperse at high ionic strength owing to the interparticle electrostatic and steric repulsion generated by the surface-grafted ssDNA, the fully matched duplex formation on the AuNP surface triggers the spontaneous rapid aggregation of the dsDNA–AuNPs in a non-cross-linking manner. One possible driving force is multiple stacking interaction
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between the terminal base pairs facing the solvent.15 This unique colloidal behavior, which has been utilized for various bioassays,16-23 is applied to the signal transduction in the Cry j 2 detection method. EXPERIMENTAL SECTION Reagents and apparatus Citrate-capped AuNP with a diameter of 15 nm was purchased from BBI (UK). Japanese cedar (Cryptomeria japonica) pollen allergens Cry j 1 and Cry j 2 were obtained from Funakoshi (Japan). The timothy (Phleum pratense) pollen allergen Phl p 5, European house dust mite (Dermatophagoides pteronyssinus) group 2 allergen Der p 2, German cockroach (Blattella germanica) allergen Bla g 4, and the dog (Canis familiaris) allergen Can f 2 were purchased from CosmoBio (Japan). Unmodified DNA strands and 5´-mercaptohexyl DNA strands were synthesized by Eurofins Genomics (Japan) and Tsukuba Oligo Service (Japan), respectively. The base sequences are depicted in Figure S1. All the other reagents were purchased from Wako Pure Chemicals (Japan). Ultrapure water (>18 Mcm) was prepared using a Milli-Q pure water purification system (Millipore, USA). Absorption spectra were recorded with a Cary 50 UV/vis spectrophotometer (Varian, USA). Fluorescence intensities were measured on a microplate reader (PerkinElmer, USA). All photographs were taken with a Redmi Note 5 smartphone (Xiaomi, China). Preparation of ssDNA–AuNPs Conjugation of ssDNA to the surface of AuNP was achieved via Au–S bond formation, according to the reported procedure.14,24 Briefly, 5´-mercaptohexyl ssDNA (5 nmol) reduced with dithiothreitol was added to an AuNP dispersion (1 mL). After the mixture was incubated at 50 °C for 40 h using 10 mM phosphate buffer (pH 7.4) containing 0.1 M NaCl, the dispersion was centrifuged at 14000 rpm for 25 min. The ssDNA–AuNPs were resuspended in 10 mM phosphate buffer (pH 7.4) containing 0.1 M NaNO3 and stored at 4 °C. The number of surface-grafted ssDNAs per particle was evaluated with the mercaptoethanol displacement method.25 Fluorescent detection of Cry j 2 using molecular beacons A mixture of 50 μg/mL Cry j 2 (2 μL), 10 μM H1 (2 μL), 10 μM H2 (2 μL), and 1× PBS buffer (4 μL) was incubated at 37 °C for 60 min. An aliquot (5 μL) of the mixture was added to an aqueous solution (95 μL) of a molecular beacon (FP1). After the obtained solution was incubated at room temperature for 5 min, the fluorescent intensity was measured using the microplate reader with excitation at 485 nm and recording emission at 535 nm. Colorimetric detection of Cry j 2 using unmodified AuNPs
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A mixture of Cry j 2 (2 μL), 80 μM H1 (2 μL), 80 μM H2 (2 μL), and 1× PBS buffer (4 μL) was incubated at 37 °C for 60 min. Separately, 10 μM P1 (1 μL), 10 μM P2 (1 μL), and ultrapure water (11 μL) were added to the dispersion of unmodified AuNPs (20 μL), followed by incubation at 37 °C for 60 min to afford P1/P2-adsorbed AuNPs. Then, an aliquot (1 μL) of the Cry j 2 recycling reaction solution was added to the dispersion of the P1/P2-adsorbed AuNPs (33 μL). The obtained mixture was incubated at room temperature for 10 min. Subsequently, 1 M NaCl (1 μL) and ultrapure water (5 μL) was further added to the mixture to give a final volume of 40 μL. After the final solution was equilibrated for 10 min, the UV/vis absorption spectrum was measured with the wavelength ranging from 400 to 800 nm. Colorimetric detection of Cry j 2 using ssDNA–AuNPs A mixture of Cry j 2 (1 μL), 50 μM H1 (2 μL), 50 μM H2 (2 μL), and 1× PBS buffer (5 μL) was incubated at 37 °C for 60 min. An aliquot (2 μL) of the mixture was added to 6 μM P1 (2 μL). After the mixture thus obtained was allowed to stand at room temperature for 10 min, an aliquot (4 μL) of the mixture was added to the dispersion of ssDNA–AuNPs (20 µL). The as-prepared dispersion was incubated at room temperature for 10 min. Finally, 5 M NaCl (12 μL) and ultrapure water (4 μL) were added to the dispersion (24 μL). After the final solution (40 μL) was equilibrated at room temperature for 5 min, the UV–vis absorption spectrum was measured with a wavelength ranging from 400 to 800 nm. For detection of Cry j 2 in the soil sample, various amounts of soil obtained from a site on the Wako campus of RIKEN were added to ultrapure water (1 mL), and briefly shaken by hand. The dispersion was then spiked with Cry j 2 before filtration with a 0.22 μm syringe filter. RESULTS AND DISCUSSION Catalytic recycling of Cry j 2 Initially, we designed a catalytic target-recycling system to endow the present assay with high sensitivity (Figure 1A). The system consists of two DNA hairpins, H1 and H2. The base sequences are depicted in Figure S1. H1 is composed of three segments: the first one working as an anti-Cry j 2 DNA aptamer,7 the second one initiating hybridization between H1 and H2, and the third one forming a DNA three-way junction (3WJ) structure. H2 contains two segments: the first one complementary to the initiation segment of H1 and the second one forming the 3WJ structure. In the presence of Cry j 2, the aptameric segment of H1 binds to Cry j 2, accompanying the opening of the stem-loop structure. The resultant disclosure of the initiation segment of H1 triggers the hybridization with H2 and the simultaneous dissociation of Cry j 2 from H1. The released Cry j 2 is captured again by H1, thereby catalytically producing the H1/H2 DNA duplex. In the absence of Cry j 2, the thermodynamically
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Figure 1. (A) Schematic illustration of the catalytic Cry j 2 recycling. (B) Schematic illustration of the red-to-purple color change caused by salt-induced aggregation of bare AuNPs generated by detachment of DNA probes (P1 and P2) from the AuNP surfaces to form two DNA three-way junctions in the presence of Cry j 2. (C) Schematic illustration of the lack of color change due to colloidal stabilization of AuNPs induced by adsorption of P1 and P2 in the absence of Cry j 2. (D) Absorption spectra and (E) the ratio of the absorbance at 610 and 520 nm of the AuNP dispersion in the presence of (1) 50 nM H1, 50 nM H2, and 500 ng/mL Cry j 2, (2) 50 nM H1 and 50 nM H2, and (3) 500 ng/mL Cry j 2. The error bars are the standard deviation of the mean from three measurements. stable H1 and H2 are not allowed to form the H1/H2 duplex under the present conditions. Therefore, the quantity of Cry j 2 is translated into the quantity of the H1/H2 duplex in an amplified manner. To verify this recycling system, we used a molecular beacon technique (Figure S2A). When Cry j 2 was added to the reaction solution containing H1, H2, and a molecular beacon probe (FP1), we observed a significant increase in fluorescent intensity (Figure S2B). This is because the H1/H2 duplex thus produced underwent opening of FP1 to reduce the fluorescence resonance energy transfer efficiency, accompanied by formation of the 3WJ structure with FP1. This result demonstrated that the recycling amplification was indeed initiated by Cry j 2 to produce the H1/H2 duplex. Colorimetric detection of Cry j 2 using unmodified AuNPs Next, we attempted to visually detect the H1/H2 duplex using colloidal aggregation of AuNPs. Prior to the use of ssDNA–AuNPs, we designed a detection scheme using unmodified AuNPs because of the greater simplicity. Figure 1B shows the scheme for the colorimetric detection of the H1/H2 duplex using unmodified AuNPs in the presence of two DNA probes (P1 and P2). When the P1/P2-
adsorbed AuNPs are supplied, the H1/H2 duplex accommodates P1 and P2, both of which are dissociated from the AuNP surfaces, to form the 3WJ structure at both ends, thereby producing the H1/H2/P1/P2 quaternary complex. In general, a stiffer sugar-phosphate backbone without exposed nucleobases significantly attenuates the physical adsorption of dsDNA onto the AuNP surface, compared to the physical adsorption of ssDNA.26 The resultant bare AuNPs undergo salt-induced aggregation with a solution color change from red to purple. In the absence of Cry j 2, neither H1 nor H2 can cause detachment of P1 and P2 from the AuNP surfaces, and thus no color change is observed (Figure 1C). To assess the feasibility of this scheme, absorption spectra of the AuNP dispersion were measured with or without Cry j 2. The plasmon band of the AuNPs was observed at around 520 nm (Figure 1D). When Cry j 2 was added to an aqueous solution of H1 and H2, the absorbance at 610 nm increased, whereas the absorbance at 520 nm was almost unchanged. In the absence of Cry j 2, the AuNP dispersion exhibited no change in the absorbance at 610 nm. Therefore, we used the ratio of the absorbance at 610 and 520 nm (A610/A520) to evaluate the degree of the AuNP aggregation. A higher ratio indicates a more advanced aggregation state. Figure 1E shows that the A610/A520 value increased by 40% in the presence of 500
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Figure 2. Analytical performance of the colorimetric Cry j 2 assay using unmodified AuNPs. (A) Absorption spectra of the AuNP dispersion at various Cry j 2 concentrations. The final concentrations of H1, H2, P1, P2, and NaCl were 400 nM, 400 nM, 500 nM, 500 nM, and 25 mM, respectively. (B) Calibration plot of Cry j 2 using the ratio of the absorbance at 610 and
520 nm of the AuNP dispersion at various Cry j 2 concentrations. The “3 line” is drawn at the zero-dose value plus 3-times standard deviation of the zero-dose measurements (n = 3). The linear relationship was observed at the Cry j 2 concentration ranging from 0.2 to 4 μg/mL (R2 = 0.989). The photographs of the AuNP dispersions containing Cry j 2 at different concentrations are also shown. (C) Absorption spectra and (D) the ratio of the absorbance at 610 and 520 nm in the presence of various allergen proteins at 500 ng/mL. The inset to (D) shows the color changes of the AuNP dispersion containing (1) no allergen protein, (2) Cry j 1, (3) Cry j 2, (4) Phl p 5, (5) Can f 2, (6) Bla g 4, and (7) Der p 2. The error bars are the standard deviation of the mean from three measurements. ng/mL Cry j 2, compared to the value without Cry j 2, demonstrating the validity of the assay. To achieve better sensitivity, we optimized various parameters including the temperature, the concentrations of H1, H2, and NaCl, the incubation time for the AuNP aggregation, and the number of mismatches that were involved in H2. For assessment of the assay performance, we calculated the signal-to-noise ratio (SNR) by comparing the A610/A520 value in the presence and absence of Cry j 2. Figure S3A shows that SNR increased with increasing temperature for the catalytic Cry j 2 recycling from 25 °C to 37 °C, but sharply decreased at 44 °C. This was probably due to the thermal denaturation of Cry j 2, which could have decreased the efficiency of molecular recognition by the aptameric segment of H1. Thus, we determined the optimal temperature to be 37 °C. Figure S3B shows that SNR increased with increasing concentrations of both H1 and H2 from 50 nM to 400 nM, and leveled off at 800 nM. Therefore, we decided to use H1 and H2 at concentrations of 400 nM each for the catalytic Cry j 2 recycling. Figure S3C shows that SNR increased slightly with increasing NaCl concentration for the AuNP aggregation from 20 mM to 25 mM, but sharply decreased from 25 mM to 30 mM. This was likely because even DNA-adsorbed AuNPs
underwent salt-induced aggregation under a high salt concentration. The largest SNR value was obtained at a concentration of 25 mM NaCl, which was used for the following experiments. As shown in Figure. S3D, SNR increased with increasing incubation time for the AuNR aggregation up to 10 min, and then leveled off. Therefore, we determined the optimal incubation time for the AuNR aggregation to be 10 min before the measurement of absorption spectra. Finally, we optimized the number of mismatches in H2. Previous studies demonstrated that the introduction of mismatched bases into DNA hairpins improved the efficiency of duplex formation between the DNA hairpins.27,28 This fact prompted us to install three mismatches in the H2 hairpin (Figure S1). For comparison, we introduced one mismatch (H2(1MM)), two mismatches (H2(2MM)), and four mismatches (H2(4MM)) into the perfect-matched version of H2 (H2(PM)). Figure S3E shows that the introduction of 1–4 mismatched base(s) indeed increased the SNR. The highest SNR value was achieved with the original three-base-mismatched hairpin (H2). Therefore, we decided to employ H2 in the following experiments. The number of mismatches had a negligibly small effect on the A610/A520 values without Cry j 2, indicating that the undesired hybridization between H1 and H2 in the absence of Cry j 2 was not accelerated even by
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Figure 3. (A) Schematic illustration of the absence of color change due to high colloidal stability of cP1–AuNPs and formation of a DNA three-way junction in the presence of Cry j 2. (B) Schematic illustration of the red-to-purple color change caused by salt-induced non-cross-linking aggregation of P1/cP1–AuNPs in the absence of Cry j 2. (C) Absorption spectra of the AuNP dispersion with or without 500 ng/mL Cry j 2.
introducing the mismatches to H2(PM) under the present conditions. Table S1 summarizes the working ranges and optimal values of the parameters. Next, we investigated the sensitivity and selectivity of the assay under the optimal conditions. The plasmon band at 520 nm was gradually red-shifted with increasing Cry j 2 concentration (Figure 2A). A linear dependence of the A610/A520 value on log[Cry j 2] was observed in the Cry j 2 concentration range from 0.2 to 4 μg/mL (5.4 to 108 nM) (Figure 2B). The limit of detection (LOD) was determined to be 0.2 μg/mL. We also examined the selectivity of the assay. For a non-target allergen protein, we used the Japanese cedar allergen Cry j 1, timothy allergen Phl p 5, dog allergen Can f 2, cockroach allergen Bla g 4, and mite allergen Der p 2. No significant change was observed in the absorption spectra when either Cry j 1, Phl p 5, Can f 2, or Bla g 4 was used; however, Der p 2 induced a remarkable red shift of the plasmon band, indicating AuNP aggregation (Figure 2C). The A610/A520 value and the solution color are depicted in Figure 2D. We hypothesized that the undesired AuNP aggregation with Der p 2 occurred owing to the physical or chemical adsorption of Der p 2 onto the AuNP surface. The three disulfide bonds displayed on the surface of Der p 229 could engage in multidentate coordination interactions with the surface Au atoms, thereby cross-linking the AuNPs. Importantly, this result implies that a colorimetric assay using unmodified AuNPs could interfere with unexpected adsorption of non-target proteins and other molecules in sample solutions. Therefore, we next refined the current assay by protecting the AuNP surface. Colorimetric detection of Cry j 2 using ssDNA–AuNPs
The schemes for the colorimetric assay using ssDNA– AuNPs are summarized in Figure 3A and B. The surfacegrafted ssDNA (cP1) was designed to be complementary to P1. In the presence of Cry j 2, the H1/H2 duplex produced by the same catalytic Cry j 2 recycling (Figure 1A) forms the 3WJ structure with P1. When the recycling reaction solution is added to the dispersion of cP1–AuNPs, no aggregation takes place because the cP1–AuNPs are stably dispersed even at high NaCl concentration (Figure 3A). By sharp contrast, in the absence of Cry j 2, the recycling reaction solution contains only H1 and H2, neither of which can be hybridized to P1. Accordingly, the addition of the recycling reaction solution to the cP1–AuNP dispersion triggers the hybridization of P1 to cP1–AuNPs, leading to the salt-induced, non-cross-linking aggregation of fully matched dsDNA–AuNPs (Figure 3B). As shown in Figure S4, we optimized the DNA grafting density and the NaCl concentration to improve the performance of the assay. The DNA grafting density of 110 strands per particle at the NaCl concentration of 1.5 M provided the largest SNR value (Figure S4A). The higher DNA density (150 strands/AuNP) offered a significantly lower SNR value, because the highly packed ssDNA brushes could sterically and electrostatically hinder the access of P1 to form the duplex on the AuNP surface. In addition, we surveyed the effect of the P1 concentration (Figure S4B). The SNR value increased with increasing concentration of P1 until 300 nM; however, a significant decrease occurred above 300 nM. Thus, we determined the optimal P1 concentration to be 300 nM. Table S2 shows the working ranges and optimal values of the parameters investigated in this study.
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Figure 4. Analytical performance of the colorimetric Cry j 2 assay using ssDNA–AuNPs. (A) Absorption spectra of the cP1– AuNP dispersion after Cry j 2 was added at various concentrations. The final concentrations of H1, H2, P1, and NaCl were 500 nM, 500 nM, 300 nM, and 1.5 M, respectively. (B) Calibration plot of Cry j 2 using the ratio of the absorbance at 530 and 610 nm of the AuNP dispersion at various Cry j 2 concentrations. The “3 line” is drawn at the zero-dose value plus 3-times standard deviation of the zero-dose measurements (n = 3). The linear relationship was observed at the Cry j 2 concentration ranging from 0.2 to 2 μg/mL (R2 = 0.959). The photographs of the AuNP dispersions containing Cry j 2 at different concentrations are also shown. (C) Absorption spectra and (D) the ratio of the absorbance at 530 and 610 nm in the presence of various allergen proteins at 500 ng/mL. The inset to (D) shows the color changes of the AuNP dispersion containing (1) no allergen protein, (2) Cry j 1, (3) Cry j 2, (4) Phl p 5, (5) Can f 2, (6) Bla g 4, and (7) Der p 2. The error bars are the standard deviation of the mean from three measurements
Figure 5. The colorimetric detection of Cry j 2 added to an aqueous soil solution filtrate using the salt-induced noncross-linking aggregation of the dsDNA–AuNPs. The error bars are the standard deviation of the mean from three measurements. Under these optimized conditions, we determined the detection limit of Cry j 2 for the assay using the ssDNA– AuNPs. Figure 4A shows that the plasmon band at 550 nm was gradually blue-shifted to 530 nm with increasing Cry j 2 concentration, while the absorbance between 600 nm
and 700 nm was concomitantly decreased. We used the ratio of the absorbance at 530 and 610 nm (A530/A610) to evaluate the degree of dispersion of the AuNPs; namely, higher A530/A610 indicated a more advanced dispersion state caused by a larger amount of Cry j 2. A good linear relationship between the A530/A610 value and log[Cry j 2] was observed at the Cry j 2 concentration ranging from 0.2 to 2 μg/mL (5.4 to 54 nM); the LOD value was determined to be 0.2 μg/mL (5.4 nM) (Figure 4B). Thus, both the linear range and LOD of the assay with the ssDNA–AuNPs were almost the same as those of the assay with the unmodified AuNPs (Figure 2B). The sensitivity of both assays was approximately 130-fold greater than that of the previously reported biosensor.7 The improved LOD was probably mainly attributable to the catalytic Cry j 2 recycling. Figure 4B also shows that the color of the dispersion was gradually changed from purple to red with increasing Cry j 2 concentration. We examined the selectivity of the assay by employing the same set of allergen proteins. Figure 4C and D show that the assay produced no response to any of the nontarget allergen proteins, whereas Cry j 2 drew an approximately twice-larger response than the blank sample. As described above, the assay using the unmodified AuNPs showed the undesired response to Der p 2. In contrast, the new assay using the ssDNA–AuNPs exhibited no response to Der p 2. Taken together, these
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results show that the colorimetric assay using the ssDNA– AuNPs showed a comparable sensitivity and greater selectivity toward Cry j 2, compared to the assay using the unmodified AuNPs. Finally, we applied the assay using the ssDNA–AuNPs to the detection of Cry j 2 in soil solution filtrates. An aqueous soil solution filtrate spiked with Cry j 2 was used as a model sample. Prior to the detection of Cry j 2, we examined the side effects of the soil solution filtrate on the current assay. When the soil solution (25 mg/mL) filtrate without Cry j 2 was used as a sample, the A530/A610 value of the soil solution filtrate was almost undistinguishable from that of the buffer solution (Figure 5 and S5). This result strongly suggested that no significant interference was caused by microorganisms and/or soil organic substances, and prompted us to use the Cry j 2-spiked soil solution filtrate as the model sample. As expected, the A530/A610 value obtained for the Cry j 2 detection in the soil solution filtrate was almost the same as that obtained using the buffer solution (Figure 5). The colorimetric assay based on the salt-induced non-cross-linking aggregation of dsDNA– AuNPs holds promise for environmental Cry j 2 monitoring.
AUTHOR INFORMATION Corresponding Authors *Email:
[email protected] (C.C. Chang);
[email protected] (T. Takarada) Phone: +8863-581-8002 (C.C. Chang); +8148-467-5489 (T. Takarada) ORCID Chia-Chen Chang: 0000-0001-5466-4724 Tohru Takarada: 0000-0001-6906-5812 Acknowledgements This work was supported by the Taiwan Postdoctoral Research Abroad Program (Grant 105-2917-I-564-034), JSPS KAKENHI Grant JP25220204, and a Grant for Molecular Systems Research provided by RIKEN. Notes The authors declare no competing financial interest.
REFERENCES
CONCLUSIONS In this study, we developed a highly sensitive colorimetric method to detect Cry j 2 based on the catalytic recycling of the target protein and the non-cross-linking aggregation of dsDNA–AuNPs. The solution color changed from purple to red in the presence of Cry j 2. The assay exhibited a good LOD that was approximately 130-fold greater than that of the previously reported assay, and showed sufficiently high selectivity toward Cry j 2. Although the unmodified AuNP also served as a colorimetric probe, the bare surface yielded a false-positive response to the non-target allergen protein, Der p 2. By contrast, the assay using the ssDNA–AuNPs produced no undesired color changes with various non-target allergen proteins and exhibited high stability in the environmental samples. These properties strongly suggest the suitability of the assay for practical use. The present methodology could be readily applied to detect various proteins by using appropriate aptamers as the recognition segment of H1 for environmental assessments and biomedical diagnostics. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssensors.xxxxxxx. Base sequences and secondary structure of the DNA components, fluorescence intensity data, sensing responses to different detection conditions (PDF)
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