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Integrating Target-Triggered Aptamer-Capped HRP@Metal−Organic Frameworks with a Colorimeter Readout for On-Site Sensitive Detection of Antibiotics Lumin Wang, Guangjuan Liu, Yuxiang Ren, Yinghui Feng, Xinyi Zhao, Yuqiu Zhu, Miao Chen, Fawei Zhu, Qi Liu,* and Xiaoqing Chen*
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ABSTRACT: Colorimetric analytical strategies exhibit great promise in developing on-site detection methods for antibiotics, while substantial recent research efforts remain problematic due to dissatisfactory sensitivity. Taking this into account, we develop a novel colorimetric sensor for in-field detection of antibiotics by using aptamer (Apt)-capped and horseradish peroxidise (HRP)embedded zeolitic metal azolate framework-7 (MAF-7) (Apt/HRP@MAF-7) as target recognition and signal transduction, respectively. With the substrate 3,3′,5,5′-tetramethylbenzidine (TMB)-impregnated chip attached on the lid, the assay can be conveniently operated in a tube and reliably quantified by a handheld colorimeter. Hydrophilic MAF-7 can not only prevent HRP aggregation but also enhance HRP activity, which would benefit its detection sensitivity. Besides, the catalytic activity of HRP@MAF-7 can be sealed through assembling with Apt and controllably released based on the bioresponsivity via forming target-Apt complexes. Consequently, a significant color signal can be observed owing to the oxidation of colorless TMB to its blue-green oxidized form oxTMB. As a proof-of-concept, portable detection of streptomycin was favorably achieved with excellent sensitivity, which is superior to most reported methods and commercial kits. The developed strategy affords a new design pattern for developing on-site antibiotics assays and immensely extends the application of enzyme embedded metal−organic framework composites.
O
Horseradish peroxidase (HRP) is a kind of brilliant signal transduction material for high-performance assaying as it could catalyze the oxidization of numerous chromogenic substrates such as 3,3′,5,5′-tetramethylbenzidine (TMB) and o-phenylenediamine in the presence of H2O2, yielding distinguishable colorimetric signals. Frequently, numerous HRP-based colorimetric assays have been constructed for food and environmental supervision.10,11 Nevertheless, the practical applications of HRP-catalyzed reactions are usually thwarted by their inherent instability and low shelf-life in harsh operational conditions.12 The immobilization of HRP in/on supports has been proven to be an efficient solution to circumvent these obstacles. Particularly, metal−organic frameworks (MOFs) have been adopted as excellent immobilization supports for HRP de novo encapsulation,13 which can not only shield the
wing to the abuse of antibiotic drugs, overdose pharmaceutical residues in food and environment have serious threat to the health of human beings and the stability of ecosystem.1 Therefore, it is imperative to develop simple and cost-effective methods for on-site determination of antibiotics, especially in resource-deficient settings. To date, multiple classical techniques have been introduced to field-based detection of antibiotics, including colorimetry, electrochemistry, surface-enhanced Raman spectroscopy, and fluorescence.1−5 Among them, colorimetric methods have captivated considerable attention due to their application in sophisticated instruments, complicated protocols, and sophisticated techniques.6−8 Accordingly, multiple color signal transduction materials such as artificial enzymes and precious metal nanostructures have been harnessed for monitoring antibiotics. However, weak catalytic activity and specificity of artificial enzymes would decrease their signal transfer efficiency. Moreover, the intrinsic instability of metal nanomaterials still impedes the accurate identification of potential antibiotics.9 Taking account of this actuality, it is highly desirable to develop sensitive colorimetric sensors with efficient and stable signal transduction modes for on-site detection of antibiotics. © XXXX American Chemical Society
Received: September 2, 2020 Accepted: September 21, 2020
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enzymes against external stimulus but also allow the selective mass transfer of the guest molecules via the accessible micropore network of the exoskeleton.14 However, the current application of HRP-embedded MOFs (HRP@MOFs) materials is limited to enzyme substrate research. Therefore, the functionalization of HRP@MOFs with target-responsive units is particularly interesting and deserves to be widely concerned. Termed as “chemical antibodies”, aptamer (Apt) is artificial single-stranded (ss) DNA/RNA which can fold into secondary or tertiary structures and make them bind to certain targets with extremely high specificity and has already found broad applications in biomedical diagnostics and environmental monitoring.15 Meanwhile, numerous recent research studies have demonstrated that the π-electron-rich ligands and partially coordinated metal ions would endow the MOF surface with well adsorption affinity for negatively charged Apt via π−π stacking and electrostatic interactions.10,16 Consequently, Apt is suitable for serving as the target recognition capping units for enzyme@MOFs to control the catalytic activity of an encapsulated enzyme. Interestingly, the conjugation of target-responsive Apt with enzyme@MOFs has not been well explored and has been limited to serving ssDNA as the fluorescence signal carrier in imaging of microRNA in living cells.17 At this stage, employing Apt functionalized HRP@MOFs (Apt/HRP@MOFs) as the target recognition section and signal transduction mode for sensitive colorimetric monitoring of antibiotics would be workable and groundbreaking. The results presented in our study highlight the first example of controlling enzyme@MOFs catalytic activity by the target-responsive Apt-functionalized mode. On the other side, a portable and reliable readout mode is also significant for developing a sensitive colorimetric sensor. Recently, portable smartphone-based sensing and imaging devices have attracted intense attention due to their capability for on-site and rapid quantification.18 However, the color balancing function of smartphones is optimized for photography in high ambient light, so the photos are easily distorted. Furthermore, the photo resolution among different smartphones is dramatically different, so the reproducibility of photo analysis is hard to guarantee. In contrast, colorimeter, a small commercialized device with built-in self-referenced system and stable measurement performance, is well suited to work as an external colorimetric readout based on red-green-blue (RGB) digital signal. Motivated by the above consideration, we herein report a colorimetric sensor for on-site sensitive detection of antibiotics, comprising a target-responsive Apt/HRP@zeolitic metal azolate framework-7 (Apt/HRP@MAF-7) probe, a TMB-based chip-modified microtube device (TMB-MD), and a handheld colorimeter. As the blueprint of the designed Apt/HRP@MAF-7 illustrated in Figure 1a, we initially embedded the HRP molecules into the hydrophilic MAF-7 support matrix under mild biocompatible conditions. Presumably, as a kind of promising MOFs with a hydrophilic skeleton, MAF-7 could not only hinder the aggregation of the HRP but also guarantee high enzymatic activity.19 Then, HRP@MAF-7 was further modified with an Apt shell through π−π stacking and electrostatic interactions, yielding Apt/HRP@MAF-7 probe. Then, in the detection cellTMB-MD, the catalytic activity of HRP@MAF-7 would be sealed and could be controllably released by the Apt-target reaction in a concentration-dependent manner. After turn over the tube, the probe could efficiently catalyze the conversion of a white
Article
Figure 1. Schematic representation of the Apt/HRP@MAF-7 fabrication procedure (a) and the principle of the colorimetric sensor for on-site sensitive detection of targets (b).
TMB-based chip into distinctly blue-green color (oxTMB) in the presence of H2O2. Finally, an RGB signal of the chip-fixed lid could be easily recorded by a handheld colorimeter (Figure 1b). By using streptomycin (STR) as a proof of concept, highly sensitive and specific detection was favorably achieved, promising the present assay a great potential in practical applications. Prominently, this proposed strategy is generic and can be extended to other antibiotics on-site monitoring by varying the Apt.
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EXPERIMENTAL SECTION Preparation of MAF-7, HRP@MAF-7, FITC-HRP@MAF7, and Apt/HRP@MAF-7 Composites. MAF-7 was prepared by previously reported with some modifications.19 2 mL of Zn(NO3)2 aqueous solution (0.0594 g) was added into 2 mL of 3-methyl-1,2,4-triazole (Hmtz) aqueous solution (0.0841 g) at 25 °C. Then, the mixture turned milky after mixing, and after ageing for about 24 h, the resulting precipitates were recovered by centrifugation at 10,000 rpm for 10 min and washed with deionized water three times. The obtained crystals were dried at 65 °C overnight. The HRP or fluorescein isothiocyanate (FITC)-labeled HRP (FHRP) was in situ embedded in MAF-7 by introducing 10 mg of HRP or FHRP into Hmtz aqueous solution during the MAF-7 preparation process, and the product was denoted as HRP@ MAF-7 (FHRP@MAF-7). The obtained precipitate was gathered by centrifuging separation at 10,000 rpm for 10 min, then washed by deionized water three times, and freezedried. For preparation of Apt/HRP@MAF-7 composites, 10 mg of HRP@MAF-7 was incubated with 5 mL of Tris−HCl buffer (pH 7.4, 10 mM) covering 20 μM Apt (the sequences of Apt are shown in Table S1) at 37 °C for 6 h. Fabrication of TMB-MD for Target Detection. The fabrication of TMB-MD for antibiotics detection: the Whatman no. 1 chromatography paper was first cut into circular discs with 0.60 cm diameter using a paper hole puncher and then soaked in TMB solution (2.5 mg/mL in DMSO) for 30 min with gentle stirring. After that, the obtained TMB-based chip was fixed on the lid of a 0.5 mL Eppendorf tube to obtain the TMB-MD, which was served as the detection cell. B
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Figure 2. SEM images of MAF-7 (a), HRP@MAF-7 (b), and Apt/HRP@MAF-7 (c). (d) XRD patterns of the pure MAF-7, HRP@MAF-7, and Apt/HRP@MAF-7 composites. (e) Zeta potentials of HRP, MAF-7, HRP@MAF-7, and Apt/HRP@MAF-7 composites. The error bars show the standard deviation for five independent experiments (error bars, SD, n = 5). (f) TGA curves of MAF-7, HRP@MAF-7, Apt/HRP@MAF-7, and HRP.
Analytical Procedure. Briefly, 90 μL of Tris−HCl buffer (10 mM, pH 6.0) and 10 μL of the as-prepared Apt/HRP@ MAF-7 (2 mg/mL) were added into 10 μL of different target solutions with varied concentrations. Then, the mixed solution was incubated for 20 min at ambient temperature. After that, 5 μL of 10 mM H2O2 solution was added into the abovementioned reaction solution, and the lid of tube was covered immediately. Subsequently, the MD was placed upside down, and 1 min later, the resulted chip was recorded with a portable colorimeter (Figure S1) and subjected for further RGB analysis. Data are presented in terms of total color differences (ΔC) using the Euclidean distance eq 120,21 ΔC =
(ΔR )2 + (ΔG)2 + (ΔB)2
with incubation time and tended to level off after 10 h (Figure S2b). We characterized the morphologies of MAF-7 and HRP@ MAF-7 using a scanning electron microscope (SEM). As the results shown in Figure 2a, the pure MAF-7 displays a standard rhombic dodecahedral structure with a uniform diameter of about 200 nm. While the morphology of HRP@MAF-7 composites becomes more spherical when compared with bare MAF-7, the average size decreases to about 185 nm owing to the aggregative growth kinetics mediated by HRP23 (Figure 2b), which is further confirmed by dynamic light scattering (DLS) (Figure S3). Additionally, X-ray diffraction (XRD) patterns were performed for MAF-7 and HRP@MAF-7 (Figure 2d). Compared with original MAF-7, the characteristic diffraction peak intensities of HRP@MAF-7 decreased significantly, as the enzyme molecules occupied a part of the sites of MAF-7 and thus degraded the crystalline integrity of the MOFs frame. Samples of the as-synthesized composites were then examined by Fourier transform infrared spectroscopy (FTIR) (Figure S4). The same peaks between 1550 and 800 cm−1 are observed in the FT-IR spectra of bare MAF-7 crystal and HRP@MAF-7 composites. Meanwhile, the amide I peak at 1644 cm−1 corresponding to the stretching modes of CO bond is present in HRP and HRP@MAF-7 composites, implying the successful encapsulation of HRP in MAF-7 crystal.11 Besides, after the negative charged HRP being embedded into the MAF-7 frame, the zeta potential of HRP@ MAF-7 became less positive (decreased from +25.0 mV of bare MAF-7 to +19.1 mV of HRP@MAF-7) (Figure 2e), also revealing that the HRP@MAF-7 composites were successfully fabricated. Then, the thermal gravimetric analysis (TGA) in N2 also confirmed the presence of HRP in the composites (Figure 2f). The first-stage decomposition of HRP@MAF-7 composites start from 200 to 300 °C, while the pure MAF-7 crystal has less weight loss during this temperature range. About 18 wt
(1)
where ΔR, ΔG, and ΔB are the changes in R, G, and B colors from blank values, respectively. The calibration curve for target was generated by the final results.
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RESULTS AND DISCUSSION Preparation and Characterization of As-Prepared Composites. By adopting a de novo embedding strategy, we first encapsulated the HRP into a uniform nanoscale MAF-7 frame.22 As the encapsulation efficiency of HRP in MAF-7 would be predominantly influenced by HRP concentration and incubation time, initially, the preparation of HRP@MAF-7 was optimized by varying the concentration of HRP from 1.0 to 7.0 mg/mL, and the encapsulation efficiency of HRP gradually increased from 79.6 to 97.7% with the increasing of the HRP concentration from 1.0 to 4.0 mg/mL and decreased over 4.0 mg/mL (Figure S2a). It may be attributed to that the nucleation sites are saturated at high enzyme concentrations and concurrently result in low encapsulation efficiency of HRP.10 Accordingly, 4 mg/mL of HRP was selected for subsequent studies, and for the incubation time of HRP in MAF-7, the encapsulation efficiency was positively correlated C
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and electrostatic interactions. The SEM results revealed the Apt/HRP@MAF-7 composites possessed a bigger size distribution than HRP@MAF-7 (Figure 2c), which agreed well with DLS results (Figure S3), and the introduction of Apt would reverse the zeta potential of the composites (from +19.1 mV of HRP@MAF-7 to −12.9 mV of Apt/HRP@MAF-7, Figure 2e) but not degrade the structure of HRP@MAF-7, as the XRD patterns of Apt/HRP@MAF-7 composites matched well with that of HRP@MAF-7 (Figure 2d). The smaller mass loss of Apt/HRP@MAF-7 at 200−300 °C further indicated the successful Apt shell ornament (Figure 2f). Besides, the surface area and pore volume of HRP@MAF-7 hybrid composites were determined to be 992.39 m2/g and 0.40 cm3/g, respectively, which were lower than that of bare MAF-7 (1203.59 m2/g and 0.51 cm3/g), indicating that embedding HRP into MAF-7 would result in decrease of the surface area and pore volume (Figures 3c and S7, and Table S2). However, the distribution of pore size calculated by the nonlocal densityfunctional theory (NLDFT) revealed that the incorporation of HRP into MAF-7 would not influence the pore diameter of the MAF-7 scaffold. After Apt shell capping, the N2 uptake amount of HRP@MAF-7 was significantly decreased (from 922.93 m2/ g of HRP@MAF-7 to 63.81 m2/g of Apt/HRP@MAF-7), and the negligible pore structure of Apt/HRP@MAF-7 indicated that Apt can effectively block the transport pathways of the MAF-7 frame and prevent the access of the substrates to the embedded HRP molecules. Enhancement Enzymatic Activity by MAF-7. As mentioned above, the signal transduction of the proposed platform stems from Apt-target reaction triggered enzyme catalytic reaction. Therefore, enzymatic activity of the encapsulated HRP is essential for the application of the HRP@MAF-7 composites. Thus, we assessed the performance of framework chemistry as an enzyme immobilization matrix. As the results shown in Figure S8a, the Soret absorption band at 402 nm (π−π*) and the iron-heme cofactor in HRP are substantially unchanged in the presence of Zn2+ ion and Hmtz, and the solid-state UV−visible spectrum of HRP@MAF-7 also almost coincides with that of HRP (Figure S8b). These results indicated that the protein secondary structure heme-binding pocket of HRP was well-preserved in Zn2+ ion, Hmtz, and MAF-7 frame, which would guarantee the enzymatic activity of the encapsulated HRP (Figure S8c).26 Notably, HRP@MAF-7 possesses much stronger tolerance toward alkaline pH, high temperature, organic solvents, and protease than free HRP, indicating that the MAF-7 frame could effectively protect the enzyme activity from inhospitable environments and thus provide the stability necessary for the HRP to promise their performance for practical application (Figure 4a,b). We next examined the catalytic activity of HRP@MAF-7 composites by using a simple and sensitive enzyme/TMB/ H2O2 reaction system (Figure 4c). As shown in Figure 4d, the decoration of TMB has negligible effect on the RGB signal of blank filter paper. Then, the signals of MAF-7/H2O2 (3) and Apt/H2O2 (4) reaction systems remain consistent with that of the TMB-based chip alone (1), indicating that bare MAF-7 or Apt had no catalytic activity for oxidation of TMB. Inversely, the signals of HRP/H2O2 (5) and HRP/MAF-7/H2O2 (6) reaction systems turn out to be positive, and the signal response of HRP/MAF-7/H2O2 (6) is even higher than that of the HRP/H2O2 (5), indicating that the support matrix MAF-7 would improve the catalytic activity of HRP. To further verify the enhancement effect of MAF-7 for the catalytic activity of
% of weight loss of the composite occurred during this stage, which could be attributed to the decomposition of HRP molecules. We then further investigated the distribution of HRP molecules in/on the MAF-7 frame. As the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) image shown in Figure S5, a band corresponding to the molecular weight of HRP (∼40 kDa) is obtained on the gel for both free HRP and HRP@MAF-7 samples (lane 1 and 3, respectively). In contrast, no obvious band is observed from the MAF-7 adsorbed with HRP (denoted as HRP-on-MAF-7) (lane 2). This result clearly demonstrated that HRP molecules of HRP@MAF-7 were embedded in the MAF-7 frame and cannot be removed by washing. Conversely, the adsorbed HRP on the external surface of MAF-7 can readily be washed away.24 Furthermore, to inquire the location of HRP in HRP@ MAF-7 composites and provide direct visible evidence of successful encapsulation of HRP in MAF-7 crystal, the fluorescent FHRP@MAF-7 composites were synthesized by adopting FHRP as the reactant and then subjected for confocal laser scanning microscopy imaging. The confocal microscopy of FHRP@MAF-7 revealed a good colocalization of HRP (green fluorescence) and MAF-7 (Figures 3a and S6).
Figure 3. (a) Confocal microscope images of FHRP@MAF-7 sample. Scale bars are 2 μm. (b) TEM image of HRP@MAF-7 composites and EDS mapping of elemental distributions for Zn and Fe. Scale bars are 200 nm. (c) TGA curves of MAF-7, HRP@MAF-7, Apt/HRP@ MAF-7, and HRP.
However, in the photographs of FHRP-on-MAF-7 (Figure S6), FHRP was slightly absorbed in the surface of MAF-7. It may be attributed to that HRP is easy to be washed out by water. These data clearly revealed that HRP molecules were well-dispersed throughout the entire MAF-7 frame, which was consistent with the results of SDS-PAGE. Besides, HRP is a ferric enzyme which contains an Fe ion in its heme group.25 The energy dispersive X-ray spectroscopy (EDS) mapping under the transmission electron microscopy (TEM) (Figure 3b) showed the existence and relatively uniform distribution of Fe element within the composite, which also indicated that the protein molecules were embedded in MAF-7. After the successful assembly of the HRP@MAF-7, we then fabricated the Apt/HRP@MAF-7 by assembling negatively charged Apt on the surface of HRP@MAF-7 via π−π stacking D
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Figure 4. (a) pH-activity profile of free HRP and HRP@MAF-7. (b) Activities of HRP and HRP@MAF-7 after thermal treatment (2 h), exposure to organic solvent (2 h), or in the presence of proteolytic agent (4 mg/mL protease, 2 h). (c) Proposed catalytic mechanism for the HRP@MAF-7/ TMB/H2O2 system. (d) (1) RGB changes of blank filter paper before or after the TMB treatment and (2−9) RGB changes of the prepared TMBbased chip before or after H2O2 (2); MAF-7, H2O2 (3); Apt, H2O2 (4); HRP, H2O2 (5); HRP/MAF-7, H2O2 (6); Apt@HRP/MAF-7, H2O2 (7); Apt@HRP/MAF-7, H2O2, STR (8); or Apt@HRP/MAF-7, H2O2, KANA (9) treatment. (Inset: Corresponding images of the chips under daylight lamp.) Experiment conditions: Apt@HRP/MAF-7: 2 mg/mL; TMB: 2.5 mM; H2O2: 10 mM; STR: 3 ng/mL; KANA: 1 μg/mL; STR-Apt@HRP/ MAF-7 incubation time: 20 min; and reaction time: 1 min. (Error bars, SD, n = 5).
4d and S10, the ΔC signals of the column (7) (Apt@HRP/ MAF-7, H2O2) are almost the same as that of TMB-based chip alone (1) and the steady-state signals are approached in 9 days, indicating that HRP was well-sealed by the AptSTR outside the HRP@MAF-7 composites and would not produce false positive results. While upon addition of STR, the colorimeter signal remarkably increased and nearly remained constant in 9 days after the specific Apt-STR reaction (column (8)), suggesting that Apt@HRP/MAF-7 possessed satisfactory stability. Furthermore, only specific Apt-target recognition yielded an obvious increase of the signal, because no signal can be observed in column (9) in which the STR was replaced by KANA, indicating that there was no cross-reactivity in this platform. Clearly, all the encouraging results suggested that the Apt shell could firmly suppress the catalytic activity of HRP@ MAF-7 and could not be degraded/released from the particle surface along with the storage time. Additionally, it could be specifically triggered by specific Apt-target recognition. Therefore, sensitive and selective detection of the proposed colorimetric sensor is feasible. Experimental Conditions Optimization. Based on the well-preserved enzymatic activity and target response capability of Apt@HRP/MAF-7, we then conceive of a potential application of the fabricated Apt@HRP/MAF-7 composites in in-field detection of antibiotics, in which the TMB-MD was employed as the portable carrier for detection. Thus, the paper material used in the texture TMB-based chip was first evaluated. Compared with other paper materials, the filter paper (Whatman chromatography paper #1) could afford a better signal response (Figure S11) for its high hydrophilic, excellent retention of fine particles, and uniform pore size distribution. Besides, Apt-target incubation time, concentrations of TMB and H2O2, and catalytic reaction time, which might govern the reliability and sensitivity of this system, were also systematically optimized. As the results shown in Figure S12a, the colorimeter signal positively correlates with the
HRP, the catalytic kinetics of HRP@MAF-7 composites were then investigated. As the results shown in Figure S9, the initial rates versus TMB and H2O2 concentrations both follow typical Michaelis−Menten behavior in a certain range of substrate concentration. By using TMB as the substrate, the Km values (calculated by the Lineweaver−Burk plot, Table S3) of HRP@ MAF-7 and free HRP are approximate with each other. Nevertheless, when H2O2 served as the substrate, the Km value of HRP@MAF-7 was 4-fold lower than that of free HRP. It is known that the lower Km value reflects a higher affinity between enzymes and substrates.27 Meanwhile, owing to the enhanced affinity between substrate and encapsulated HRP, the Vmax values of HRP@MAF-7 composites are higher than that of free HRP. These results confirmed that the catalytic activity of HRP@MAF-7 composites was higher than that of free HRP, which would be contributed to the following reasons: (1) the guest molecule H2O2 (1.5 Å) is easy to diffuse through the pores of HRP@MAF-7 and the MAF-7 cavity confinement effect can build up local concentration of H2O2, accompanied by the synergy of the MAF-7 shell and incorporated enzyme,28 (2) the diameter and hydrophilic properties of pores in MAF-7 frame can achieve the catalytic selectivity that enable selective and fast diffusion of reaction reagents through the interior cavities, especially for reactions in liquid phase,29 (3) the structural stability and porosity of MAF7 crystals can reduce undesirable aggregation and atomic leaching of enzyme,30 and (4) the possible released Zn2+ ion seems to activate catalytic activity of HRP as observed in other enzymes (Figure S8c).31 Logically, the enhanced catalytic activity would benefit the sensitivity of HRP@MAF-7 based detections. Feasibility Investigation. Since the enzymatic activity of the encapsulated HRP was well preserved and even improved by MAF-7 frame, we then further investigated the shielding function, stability, and target response capability of the Apt shell out of the HRP@MAF-7 composites. As shown in Figures E
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Figure 5. (a) Calibration curve for STR detection (0, 0.005, 0.05, 0.1, 0.3, 0.5, 1, 3, and 6 ng/mL). (Inset shows photographs for visible detection of STR). (b) Selectivity of the detection of STR. The STR concentration is 2 ng/mL, and others are 1 μg/mL. (c) Calibration curve of KANA with concentrations of 0, 0.5, 1, 3, 5, 10, 30, 50, 70, 100, 200, and 300 pg/mL. (Inset shows photographs for visible detection of KANA). Experiment conditions: Apt@HRP/MAF-7: 2 mg/mL; TMB: 2.5 mM; H2O2: 10 mM; Target-Apt@HRP/MAF-7 incubation time: 20 min; and reaction time: 1 min. (Error bars, SD, n = 5).
Table 1. Comparison of Assay Results of the Developed Method and Commercial ELISA Kits for Detection of STR in Real Samples (n = 5)a this method sample raw milk
Xiangjiang river
ELISA kits
spiked (ng/mL)
found (ng/mL)
recovery ± RSD (%)
found (ng/mL)
recovery ± RSD (%)
0 1 3 5 0 1 3 5
0.95 2.97 4.91 1.03 3.08 5.07
95 ± 2.0 99 ± 2.3 98.2 ± 1.5 103 ± 1.6 102.7 ± 3.1 101.4 ± 1.8
3.13 5.08 3.12 5.11
104.3 ± 4.1 101.6 ± 2.4 104 ± 3.7 102.2 ± 2.8
“” means not found.
a
μg/mL). As the results illustrated in Figure 5b, a significant ΔC enhancement is obtained for the target STR (2 ng/mL), while negligible signal responses could be observed for other antibiotics, indicating that our detection possesses satisfactory specificity and selectivity. Besides, the reproducibility and stability of the proposed method were also evaluated. By testing STR samples with three concentrations (0.3, 0.5, and 1 ng/mL), the relative standard deviation (RSD) of the intraassay and inter-assay is calculated to be 1.49−4.45% (n = 5) and 4.43−5.78% (n = 5), respectively (Table S6), demonstrating the excellent reproducibility of the detection. Furthermore, the stability of this sensor was evaluated into two groups: one was stored in the room temperature and another was kept in 4 °C (in a refrigerator). As shown in Figure S13, the detection performances of two groups are all well preserved in 7 days but steadily decrease after 15 days for the room temperature group, and the refrigeration would extend the validity of this sensor to 55 days or even longer. These results indicated that this colorimetric sensor, especially for the one being refrigerated, exhibits considerable stability. Real Sample Analysis. To assess the practicability of this sensor, raw milk (obtained from a local market) and water from the Xiangjiang river with pretreatment spiked with different concentrations (1, 3, and 5 ng/mL) of STR were subjected for proposed method and ELISA kits, respectively. As the results shown in Table 1, favorable recoveries of ranged from 95 to 103% (n = 5) and accuracy were obtained for our method, and the developed method exhibited higher sensitivity than commercial STR ELISA kits. Therefore, the considerable performance of this portable platform would provide a
incubation time from 1 to 30 min and reaches a plateau after 20 min, indicating that the specific Apt-target reaction can be essentially completed in 20 min, while the signal also increases with the addition of TMB content and reaches a balance after 2.5 mM (Figure S12b), and for the concentration of H2O2, the ΔC signal increases with the increasing concentration of H2O2 but decreases upon the concentration over 10 mM (Figure S12c), which might be attributed to the inhibition of enzyme activity under H2O2 concentrations higher over 10 mM.32 Additionally, the enzyme-catalyzed reaction can reach equilibrium within 1 min (Figure S12d). Performance Evaluation. Under these optimized conditions, the analytical performance of this colorimetric sensor was then evaluated by adopting STR as the proof-of-concept target. As shown in Figure 5a, the signal response (ΔC value) of testing dot increases linearly with the increasing concentration of STR over the range of 0.005−6 ng/mL, and the LOD is estimated to be 0.51 pg/mL, which is significantly lower than that of existed point-of-care testing platforms (Table S4) and several commercial STR ELISA kits (e.g., R-Biopharm, 5 ppb; RESEARCH AREA, 2 ppb). Meanwhile, the favorable LOD is also superior to many reported laboratory methods (Table S5). In Table S5, although the LODs of some references are lower than that of our work, complicated probe preparation procedures, high-cost, or low mobility would prevent their application in antibiotics on-site monitoring. Remarkably, the proposed method is more portable, simple, and user-friendly than many traditional and commercial detection means. To verify the selectivity of this sensor in detecting STR, we challenged our detection by using other similar antibiotics (1 F
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Xiaoqing Chen − College of Chemistry and Chemical Engineering and Key Laboratory of Hunan Province for Water Environment and Agriculture Product Safety, Central South University, Changsha 410083, Hunan, China; orcid.org/ 0000-0002-8768-8965; Email: [email protected]
promising application for on-site sensitive detection of STR in food and environment safety monitoring. Universality and Expansibility of the Developed Platform. It is noticed that the antibiotics residues are multitudinous, and the universality of sensing platform is more demanding than ever before. Thus, we generalized the proposed detection method to other antibiotics by simple varying the Apt sequence. Herein, a common aminoglycoside KANA was selected as another target contamination.33 With a similar detection strategy, the ΔC intensity was recorded in the presence of different concentrations of KANA standard solution. As the results shown in Figure 5c, a good correlation between the concentration of the KANA and ΔC response can be obtained in relatively wide dynamic ranges (0.5−300 pg/ mL). The LOD was estimated as 0.34 pg/mL, which could be comparable to other reported methods (Tables S4 and S5). The considerable universality and expansibility of this platform would promise its great application in field-based detection of various antibiotics.
Authors
Lumin Wang − College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, Hunan, China Guangjuan Liu − College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, Hunan, China Yuxiang Ren − College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, Fujian, China Yinghui Feng − College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, Hunan, China Xinyi Zhao − College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, Hunan, China Yuqiu Zhu − College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, Hunan, China Miao Chen − College of Chemistry and Chemical Engineering and School of Life Science, Central South University, Changsha 410083, Hunan, China Fawei Zhu − College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, Hunan, China Complete contact information is available at: https://pubs.acs.org/10.1021/acs.analchem.0c03723
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CONCLUSIONS In summary, a sensitive colorimetric sensor has been favorably developed for on-site detection of antibiotics by employing Apt/HRP@MAF-7 composites as the target identification and signal transduction mode and the color change of TMB-based chip as the signal reporter via portable colorimeter readout. This assay can be easily operated in a tube. We demonstrated that the MAF-7 skeleton can not only offer excellent protection to HRP but also prominently enhance enzyme catalytic activity, which is beneficial for improving the sensitivity of detection. Particularly, we find that the Apt shell capped over the surface of MAF-7 can control the catalytic activity of HRP@MAF-7, guaranteeing the selectivity of this sensor by specific Apt-target recognition reaction. Moreover, the colorimeter is free from the influence of subjective interpretation of operator and external circumstances noise, endowing the developed sensor with excellent accuracy of detection. Significantly, this strategy can be easily generalized to on-site detection of other contaminations by simply altering the corresponding Apt sequence. On the basis of easy operation, high sensitivity, and versatility, the proposed platform paves a bright route for application of enzyme@MOFs composites and holds great potential for on-site detection of trace targets in environmental inspection, food safety/drug residue inspection, and health supervision..
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge the financial support from the National Natural Science Foundation of China (21878339 & 21904141). This work was supported by The Key R&D Project in the industrial field funded by Hunan Provincial Science &Technology Department, China (2019GK2131).
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REFERENCES
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ASSOCIATED CONTENT
* Supporting Information sı
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.0c03723. Reagents and materials, instruments, determination of enzyme concentration and encapsulation efficiency, activity assay, kinetic parameters, preparation of HRPon-MAF-7 and FHRP-on-MAF-7, SDS-PAGE analysis, pretreatment of real samples, detailed figures, tables, and references (PDF)
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AUTHOR INFORMATION
Corresponding Authors
Qi Liu − College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, Hunan, China; Email: [email protected] G
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(12) Lian, X.; Fang, Y.; Joseph, E.; Wang, Q.; Li, J.; Banerjee, S.; Lollar, C.; Wang, X.; Zhou, H.-C. Chem. Soc. Rev. 2017, 46, 3386− 3401. (13) Huang, S.; Kou, X.; Shen, J.; Chen, G.; Ouyang, G. Angew. Chem., Int. Ed. 2020, 59, 8786−8798. (14) Gkaniatsou, E.; Sicard, C.; Ricoux, R.; Benahmed, L.; Bourdreux, F.; Zhang, Q.; Serre, C.; Mahy, J. P.; Steunou, N. Angew. Chem., Int. Ed. 2018, 130, 16373−16378. (15) Li, Z.; Zhang, S.; Yu, T.; Dai, Z.; Wei, Q. Anal. Chem. 2019, 91, 10448−10457. (16) Zhang, H.-T.; Zhang, J.-W.; Huang, G.; Du, Z.-Y.; Jiang, H.-L. Chem. Commun. 2014, 50, 12069−12072. (17) Xiong, M.; Zhang, M.; Liu, Q.; Yang, C.; Xie, Q.; Ke, G.; Meng, H.-M.; Zhang, X.-B.; Tan, W. Chem. Commun. 2020, 56, 2901−2904. (18) Zeng, X.; Hu, J.; Zhang, M.; Wang, F.; Wu, L.; Hou, X. Anal. Chem. 2020, 92, 2097−2102. (19) Liang, W.; Xu, H.; Carraro, F.; Maddigan, N. K.; Li, Q.; Bell, S. G.; Huang, D. M.; Tarzia, A.; Solomon, M. B.; Amenitsch, H.; Vaccari, L.; Sumby, C. J.; Falcaro, P.; Doonan, C. J. J. Am. Chem. Soc. 2019, 141, 2348−2355. (20) Soga, T.; Jimbo, Y.; Suzuki, K.; Citterio, D. Anal. Chem. 2013, 85, 8973−8978. (21) Greenawald, L. A.; Boss, G. R.; Snyder, J. L.; Reeder, A.; Bell, S. ACS Sensors 2017, 2, 1458−1466. (22) Shieh, F.-K.; Wang, S.-C.; Yen, C.-I.; Wu, C.-C.; Dutta, S.; Chou, L.-Y.; Morabito, J. V.; Hu, P.; Hsu, M.-H.; Wu, K. C.-W.; Tsung, C.-K. J. Am. Chem. Soc. 2015, 137, 4276−4279. (23) Somturk, B.; Hancer, M.; Ocsoy, I.; Ö zdemir, N. Dalton Trans. 2015, 44, 13845−13852. (24) Wu, X.; Yang, C.; Ge, J.; Liu, Z. Nanoscale 2015, 7, 18883− 18886. (25) Berglund, G. I.; Carlsson, G. H.; Smith, A. T.; Szöke, H.; Henriksen, A.; Hajdu, J. Nature 2002, 417, 463−468. (26) Dong, X.; Fan, Y.; Yang, P.; Kong, J.; Li, D.; Miao, J.; Hua, S.; Hu, C. Appl. Spectrosc. 2016, 70, 1851−1860. (27) Dong, Y.-l.; Zhang, H.-g.; Rahman, Z. U.; Su, L.; Chen, X.-j.; Hu, J.; Chen, X.-g. Nanoscale 2012, 4, 3969−3976. (28) Wu, X.; Hou, M.; Ge, J. Catal. Sci. Technol. 2015, 5, 5077− 5085. (29) Zhao, Y.; Ni, X.; Ye, S.; Gu, Z.-G.; Li, Y.; Ngai, T. Langmuir 2020, 36, 2037−2043. (30) Li, G.; Zhao, S.; Zhang, Y.; Tang, Z. Adv. Mater. 2018, 30, 1800702. (31) Lyu, F.; Zhang, Y.; Zare, R. N.; Ge, J.; Liu, Z. Nano Lett. 2014, 14, 5761−5765. (32) Gao, L.; Zhuang, J.; Nie, L.; Zhang, J.; Zhang, Y.; Gu, N.; Wang, T.; Feng, J.; Yang, D.; Perrett, S.; Yan, X. Nat. Nanotechnol. 2007, 2, 577−583. (33) Wang, L.; Zhu, F.; Chen, M.; Zhu, Y.; Xiao, J.; Yang, H.; Chen, X. Food Chem. 2019, 271, 581−587.
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Integrating Target-Triggered Aptamer-Capped HRP@Metal−Organic Frameworks with a Colorimeter Readout for On-Site Sensitive Detection of Antibiotics Lumin Wang, Guangjuan Liu, Yuxiang Ren, Yinghui Feng, Xinyi Zhao, Yuqiu Zhu, Miao Chen, Fawei Zhu, Qi Liu,* and Xiaoqing Chen*
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ABSTRACT: Colorimetric analytical strategies exhibit great promise in developing on-site detection methods for antibiotics, while substantial recent research efforts remain problematic due to dissatisfactory sensitivity. Taking this into account, we develop a novel colorimetric sensor for in-field detection of antibiotics by using aptamer (Apt)-capped and horseradish peroxidise (HRP)embedded zeolitic metal azolate framework-7 (MAF-7) (Apt/HRP@MAF-7) as target recognition and signal transduction, respectively. With the substrate 3,3′,5,5′-tetramethylbenzidine (TMB)-impregnated chip attached on the lid, the assay can be conveniently operated in a tube and reliably quantified by a handheld colorimeter. Hydrophilic MAF-7 can not only prevent HRP aggregation but also enhance HRP activity, which would benefit its detection sensitivity. Besides, the catalytic activity of HRP@MAF-7 can be sealed through assembling with Apt and controllably released based on the bioresponsivity via forming target-Apt complexes. Consequently, a significant color signal can be observed owing to the oxidation of colorless TMB to its blue-green oxidized form oxTMB. As a proof-of-concept, portable detection of streptomycin was favorably achieved with excellent sensitivity, which is superior to most reported methods and commercial kits. The developed strategy affords a new design pattern for developing on-site antibiotics assays and immensely extends the application of enzyme embedded metal−organic framework composites.
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Horseradish peroxidase (HRP) is a kind of brilliant signal transduction material for high-performance assaying as it could catalyze the oxidization of numerous chromogenic substrates such as 3,3′,5,5′-tetramethylbenzidine (TMB) and o-phenylenediamine in the presence of H2O2, yielding distinguishable colorimetric signals. Frequently, numerous HRP-based colorimetric assays have been constructed for food and environmental supervision.10,11 Nevertheless, the practical applications of HRP-catalyzed reactions are usually thwarted by their inherent instability and low shelf-life in harsh operational conditions.12 The immobilization of HRP in/on supports has been proven to be an efficient solution to circumvent these obstacles. Particularly, metal−organic frameworks (MOFs) have been adopted as excellent immobilization supports for HRP de novo encapsulation,13 which can not only shield the
wing to the abuse of antibiotic drugs, overdose pharmaceutical residues in food and environment have serious threat to the health of human beings and the stability of ecosystem.1 Therefore, it is imperative to develop simple and cost-effective methods for on-site determination of antibiotics, especially in resource-deficient settings. To date, multiple classical techniques have been introduced to field-based detection of antibiotics, including colorimetry, electrochemistry, surface-enhanced Raman spectroscopy, and fluorescence.1−5 Among them, colorimetric methods have captivated considerable attention due to their application in sophisticated instruments, complicated protocols, and sophisticated techniques.6−8 Accordingly, multiple color signal transduction materials such as artificial enzymes and precious metal nanostructures have been harnessed for monitoring antibiotics. However, weak catalytic activity and specificity of artificial enzymes would decrease their signal transfer efficiency. Moreover, the intrinsic instability of metal nanomaterials still impedes the accurate identification of potential antibiotics.9 Taking account of this actuality, it is highly desirable to develop sensitive colorimetric sensors with efficient and stable signal transduction modes for on-site detection of antibiotics. © XXXX American Chemical Society
Received: September 2, 2020 Accepted: September 21, 2020
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enzymes against external stimulus but also allow the selective mass transfer of the guest molecules via the accessible micropore network of the exoskeleton.14 However, the current application of HRP-embedded MOFs (HRP@MOFs) materials is limited to enzyme substrate research. Therefore, the functionalization of HRP@MOFs with target-responsive units is particularly interesting and deserves to be widely concerned. Termed as “chemical antibodies”, aptamer (Apt) is artificial single-stranded (ss) DNA/RNA which can fold into secondary or tertiary structures and make them bind to certain targets with extremely high specificity and has already found broad applications in biomedical diagnostics and environmental monitoring.15 Meanwhile, numerous recent research studies have demonstrated that the π-electron-rich ligands and partially coordinated metal ions would endow the MOF surface with well adsorption affinity for negatively charged Apt via π−π stacking and electrostatic interactions.10,16 Consequently, Apt is suitable for serving as the target recognition capping units for enzyme@MOFs to control the catalytic activity of an encapsulated enzyme. Interestingly, the conjugation of target-responsive Apt with enzyme@MOFs has not been well explored and has been limited to serving ssDNA as the fluorescence signal carrier in imaging of microRNA in living cells.17 At this stage, employing Apt functionalized HRP@MOFs (Apt/HRP@MOFs) as the target recognition section and signal transduction mode for sensitive colorimetric monitoring of antibiotics would be workable and groundbreaking. The results presented in our study highlight the first example of controlling enzyme@MOFs catalytic activity by the target-responsive Apt-functionalized mode. On the other side, a portable and reliable readout mode is also significant for developing a sensitive colorimetric sensor. Recently, portable smartphone-based sensing and imaging devices have attracted intense attention due to their capability for on-site and rapid quantification.18 However, the color balancing function of smartphones is optimized for photography in high ambient light, so the photos are easily distorted. Furthermore, the photo resolution among different smartphones is dramatically different, so the reproducibility of photo analysis is hard to guarantee. In contrast, colorimeter, a small commercialized device with built-in self-referenced system and stable measurement performance, is well suited to work as an external colorimetric readout based on red-green-blue (RGB) digital signal. Motivated by the above consideration, we herein report a colorimetric sensor for on-site sensitive detection of antibiotics, comprising a target-responsive Apt/HRP@zeolitic metal azolate framework-7 (Apt/HRP@MAF-7) probe, a TMB-based chip-modified microtube device (TMB-MD), and a handheld colorimeter. As the blueprint of the designed Apt/HRP@MAF-7 illustrated in Figure 1a, we initially embedded the HRP molecules into the hydrophilic MAF-7 support matrix under mild biocompatible conditions. Presumably, as a kind of promising MOFs with a hydrophilic skeleton, MAF-7 could not only hinder the aggregation of the HRP but also guarantee high enzymatic activity.19 Then, HRP@MAF-7 was further modified with an Apt shell through π−π stacking and electrostatic interactions, yielding Apt/HRP@MAF-7 probe. Then, in the detection cellTMB-MD, the catalytic activity of HRP@MAF-7 would be sealed and could be controllably released by the Apt-target reaction in a concentration-dependent manner. After turn over the tube, the probe could efficiently catalyze the conversion of a white
Article
Figure 1. Schematic representation of the Apt/HRP@MAF-7 fabrication procedure (a) and the principle of the colorimetric sensor for on-site sensitive detection of targets (b).
TMB-based chip into distinctly blue-green color (oxTMB) in the presence of H2O2. Finally, an RGB signal of the chip-fixed lid could be easily recorded by a handheld colorimeter (Figure 1b). By using streptomycin (STR) as a proof of concept, highly sensitive and specific detection was favorably achieved, promising the present assay a great potential in practical applications. Prominently, this proposed strategy is generic and can be extended to other antibiotics on-site monitoring by varying the Apt.
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EXPERIMENTAL SECTION Preparation of MAF-7, HRP@MAF-7, FITC-HRP@MAF7, and Apt/HRP@MAF-7 Composites. MAF-7 was prepared by previously reported with some modifications.19 2 mL of Zn(NO3)2 aqueous solution (0.0594 g) was added into 2 mL of 3-methyl-1,2,4-triazole (Hmtz) aqueous solution (0.0841 g) at 25 °C. Then, the mixture turned milky after mixing, and after ageing for about 24 h, the resulting precipitates were recovered by centrifugation at 10,000 rpm for 10 min and washed with deionized water three times. The obtained crystals were dried at 65 °C overnight. The HRP or fluorescein isothiocyanate (FITC)-labeled HRP (FHRP) was in situ embedded in MAF-7 by introducing 10 mg of HRP or FHRP into Hmtz aqueous solution during the MAF-7 preparation process, and the product was denoted as HRP@ MAF-7 (FHRP@MAF-7). The obtained precipitate was gathered by centrifuging separation at 10,000 rpm for 10 min, then washed by deionized water three times, and freezedried. For preparation of Apt/HRP@MAF-7 composites, 10 mg of HRP@MAF-7 was incubated with 5 mL of Tris−HCl buffer (pH 7.4, 10 mM) covering 20 μM Apt (the sequences of Apt are shown in Table S1) at 37 °C for 6 h. Fabrication of TMB-MD for Target Detection. The fabrication of TMB-MD for antibiotics detection: the Whatman no. 1 chromatography paper was first cut into circular discs with 0.60 cm diameter using a paper hole puncher and then soaked in TMB solution (2.5 mg/mL in DMSO) for 30 min with gentle stirring. After that, the obtained TMB-based chip was fixed on the lid of a 0.5 mL Eppendorf tube to obtain the TMB-MD, which was served as the detection cell. B
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Figure 2. SEM images of MAF-7 (a), HRP@MAF-7 (b), and Apt/HRP@MAF-7 (c). (d) XRD patterns of the pure MAF-7, HRP@MAF-7, and Apt/HRP@MAF-7 composites. (e) Zeta potentials of HRP, MAF-7, HRP@MAF-7, and Apt/HRP@MAF-7 composites. The error bars show the standard deviation for five independent experiments (error bars, SD, n = 5). (f) TGA curves of MAF-7, HRP@MAF-7, Apt/HRP@MAF-7, and HRP.
Analytical Procedure. Briefly, 90 μL of Tris−HCl buffer (10 mM, pH 6.0) and 10 μL of the as-prepared Apt/HRP@ MAF-7 (2 mg/mL) were added into 10 μL of different target solutions with varied concentrations. Then, the mixed solution was incubated for 20 min at ambient temperature. After that, 5 μL of 10 mM H2O2 solution was added into the abovementioned reaction solution, and the lid of tube was covered immediately. Subsequently, the MD was placed upside down, and 1 min later, the resulted chip was recorded with a portable colorimeter (Figure S1) and subjected for further RGB analysis. Data are presented in terms of total color differences (ΔC) using the Euclidean distance eq 120,21 ΔC =
(ΔR )2 + (ΔG)2 + (ΔB)2
with incubation time and tended to level off after 10 h (Figure S2b). We characterized the morphologies of MAF-7 and HRP@ MAF-7 using a scanning electron microscope (SEM). As the results shown in Figure 2a, the pure MAF-7 displays a standard rhombic dodecahedral structure with a uniform diameter of about 200 nm. While the morphology of HRP@MAF-7 composites becomes more spherical when compared with bare MAF-7, the average size decreases to about 185 nm owing to the aggregative growth kinetics mediated by HRP23 (Figure 2b), which is further confirmed by dynamic light scattering (DLS) (Figure S3). Additionally, X-ray diffraction (XRD) patterns were performed for MAF-7 and HRP@MAF-7 (Figure 2d). Compared with original MAF-7, the characteristic diffraction peak intensities of HRP@MAF-7 decreased significantly, as the enzyme molecules occupied a part of the sites of MAF-7 and thus degraded the crystalline integrity of the MOFs frame. Samples of the as-synthesized composites were then examined by Fourier transform infrared spectroscopy (FTIR) (Figure S4). The same peaks between 1550 and 800 cm−1 are observed in the FT-IR spectra of bare MAF-7 crystal and HRP@MAF-7 composites. Meanwhile, the amide I peak at 1644 cm−1 corresponding to the stretching modes of CO bond is present in HRP and HRP@MAF-7 composites, implying the successful encapsulation of HRP in MAF-7 crystal.11 Besides, after the negative charged HRP being embedded into the MAF-7 frame, the zeta potential of HRP@ MAF-7 became less positive (decreased from +25.0 mV of bare MAF-7 to +19.1 mV of HRP@MAF-7) (Figure 2e), also revealing that the HRP@MAF-7 composites were successfully fabricated. Then, the thermal gravimetric analysis (TGA) in N2 also confirmed the presence of HRP in the composites (Figure 2f). The first-stage decomposition of HRP@MAF-7 composites start from 200 to 300 °C, while the pure MAF-7 crystal has less weight loss during this temperature range. About 18 wt
(1)
where ΔR, ΔG, and ΔB are the changes in R, G, and B colors from blank values, respectively. The calibration curve for target was generated by the final results.
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RESULTS AND DISCUSSION Preparation and Characterization of As-Prepared Composites. By adopting a de novo embedding strategy, we first encapsulated the HRP into a uniform nanoscale MAF-7 frame.22 As the encapsulation efficiency of HRP in MAF-7 would be predominantly influenced by HRP concentration and incubation time, initially, the preparation of HRP@MAF-7 was optimized by varying the concentration of HRP from 1.0 to 7.0 mg/mL, and the encapsulation efficiency of HRP gradually increased from 79.6 to 97.7% with the increasing of the HRP concentration from 1.0 to 4.0 mg/mL and decreased over 4.0 mg/mL (Figure S2a). It may be attributed to that the nucleation sites are saturated at high enzyme concentrations and concurrently result in low encapsulation efficiency of HRP.10 Accordingly, 4 mg/mL of HRP was selected for subsequent studies, and for the incubation time of HRP in MAF-7, the encapsulation efficiency was positively correlated C
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and electrostatic interactions. The SEM results revealed the Apt/HRP@MAF-7 composites possessed a bigger size distribution than HRP@MAF-7 (Figure 2c), which agreed well with DLS results (Figure S3), and the introduction of Apt would reverse the zeta potential of the composites (from +19.1 mV of HRP@MAF-7 to −12.9 mV of Apt/HRP@MAF-7, Figure 2e) but not degrade the structure of HRP@MAF-7, as the XRD patterns of Apt/HRP@MAF-7 composites matched well with that of HRP@MAF-7 (Figure 2d). The smaller mass loss of Apt/HRP@MAF-7 at 200−300 °C further indicated the successful Apt shell ornament (Figure 2f). Besides, the surface area and pore volume of HRP@MAF-7 hybrid composites were determined to be 992.39 m2/g and 0.40 cm3/g, respectively, which were lower than that of bare MAF-7 (1203.59 m2/g and 0.51 cm3/g), indicating that embedding HRP into MAF-7 would result in decrease of the surface area and pore volume (Figures 3c and S7, and Table S2). However, the distribution of pore size calculated by the nonlocal densityfunctional theory (NLDFT) revealed that the incorporation of HRP into MAF-7 would not influence the pore diameter of the MAF-7 scaffold. After Apt shell capping, the N2 uptake amount of HRP@MAF-7 was significantly decreased (from 922.93 m2/ g of HRP@MAF-7 to 63.81 m2/g of Apt/HRP@MAF-7), and the negligible pore structure of Apt/HRP@MAF-7 indicated that Apt can effectively block the transport pathways of the MAF-7 frame and prevent the access of the substrates to the embedded HRP molecules. Enhancement Enzymatic Activity by MAF-7. As mentioned above, the signal transduction of the proposed platform stems from Apt-target reaction triggered enzyme catalytic reaction. Therefore, enzymatic activity of the encapsulated HRP is essential for the application of the HRP@MAF-7 composites. Thus, we assessed the performance of framework chemistry as an enzyme immobilization matrix. As the results shown in Figure S8a, the Soret absorption band at 402 nm (π−π*) and the iron-heme cofactor in HRP are substantially unchanged in the presence of Zn2+ ion and Hmtz, and the solid-state UV−visible spectrum of HRP@MAF-7 also almost coincides with that of HRP (Figure S8b). These results indicated that the protein secondary structure heme-binding pocket of HRP was well-preserved in Zn2+ ion, Hmtz, and MAF-7 frame, which would guarantee the enzymatic activity of the encapsulated HRP (Figure S8c).26 Notably, HRP@MAF-7 possesses much stronger tolerance toward alkaline pH, high temperature, organic solvents, and protease than free HRP, indicating that the MAF-7 frame could effectively protect the enzyme activity from inhospitable environments and thus provide the stability necessary for the HRP to promise their performance for practical application (Figure 4a,b). We next examined the catalytic activity of HRP@MAF-7 composites by using a simple and sensitive enzyme/TMB/ H2O2 reaction system (Figure 4c). As shown in Figure 4d, the decoration of TMB has negligible effect on the RGB signal of blank filter paper. Then, the signals of MAF-7/H2O2 (3) and Apt/H2O2 (4) reaction systems remain consistent with that of the TMB-based chip alone (1), indicating that bare MAF-7 or Apt had no catalytic activity for oxidation of TMB. Inversely, the signals of HRP/H2O2 (5) and HRP/MAF-7/H2O2 (6) reaction systems turn out to be positive, and the signal response of HRP/MAF-7/H2O2 (6) is even higher than that of the HRP/H2O2 (5), indicating that the support matrix MAF-7 would improve the catalytic activity of HRP. To further verify the enhancement effect of MAF-7 for the catalytic activity of
% of weight loss of the composite occurred during this stage, which could be attributed to the decomposition of HRP molecules. We then further investigated the distribution of HRP molecules in/on the MAF-7 frame. As the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) image shown in Figure S5, a band corresponding to the molecular weight of HRP (∼40 kDa) is obtained on the gel for both free HRP and HRP@MAF-7 samples (lane 1 and 3, respectively). In contrast, no obvious band is observed from the MAF-7 adsorbed with HRP (denoted as HRP-on-MAF-7) (lane 2). This result clearly demonstrated that HRP molecules of HRP@MAF-7 were embedded in the MAF-7 frame and cannot be removed by washing. Conversely, the adsorbed HRP on the external surface of MAF-7 can readily be washed away.24 Furthermore, to inquire the location of HRP in HRP@ MAF-7 composites and provide direct visible evidence of successful encapsulation of HRP in MAF-7 crystal, the fluorescent FHRP@MAF-7 composites were synthesized by adopting FHRP as the reactant and then subjected for confocal laser scanning microscopy imaging. The confocal microscopy of FHRP@MAF-7 revealed a good colocalization of HRP (green fluorescence) and MAF-7 (Figures 3a and S6).
Figure 3. (a) Confocal microscope images of FHRP@MAF-7 sample. Scale bars are 2 μm. (b) TEM image of HRP@MAF-7 composites and EDS mapping of elemental distributions for Zn and Fe. Scale bars are 200 nm. (c) TGA curves of MAF-7, HRP@MAF-7, Apt/HRP@ MAF-7, and HRP.
However, in the photographs of FHRP-on-MAF-7 (Figure S6), FHRP was slightly absorbed in the surface of MAF-7. It may be attributed to that HRP is easy to be washed out by water. These data clearly revealed that HRP molecules were well-dispersed throughout the entire MAF-7 frame, which was consistent with the results of SDS-PAGE. Besides, HRP is a ferric enzyme which contains an Fe ion in its heme group.25 The energy dispersive X-ray spectroscopy (EDS) mapping under the transmission electron microscopy (TEM) (Figure 3b) showed the existence and relatively uniform distribution of Fe element within the composite, which also indicated that the protein molecules were embedded in MAF-7. After the successful assembly of the HRP@MAF-7, we then fabricated the Apt/HRP@MAF-7 by assembling negatively charged Apt on the surface of HRP@MAF-7 via π−π stacking D
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Figure 4. (a) pH-activity profile of free HRP and HRP@MAF-7. (b) Activities of HRP and HRP@MAF-7 after thermal treatment (2 h), exposure to organic solvent (2 h), or in the presence of proteolytic agent (4 mg/mL protease, 2 h). (c) Proposed catalytic mechanism for the HRP@MAF-7/ TMB/H2O2 system. (d) (1) RGB changes of blank filter paper before or after the TMB treatment and (2−9) RGB changes of the prepared TMBbased chip before or after H2O2 (2); MAF-7, H2O2 (3); Apt, H2O2 (4); HRP, H2O2 (5); HRP/MAF-7, H2O2 (6); Apt@HRP/MAF-7, H2O2 (7); Apt@HRP/MAF-7, H2O2, STR (8); or Apt@HRP/MAF-7, H2O2, KANA (9) treatment. (Inset: Corresponding images of the chips under daylight lamp.) Experiment conditions: Apt@HRP/MAF-7: 2 mg/mL; TMB: 2.5 mM; H2O2: 10 mM; STR: 3 ng/mL; KANA: 1 μg/mL; STR-Apt@HRP/ MAF-7 incubation time: 20 min; and reaction time: 1 min. (Error bars, SD, n = 5).
4d and S10, the ΔC signals of the column (7) (Apt@HRP/ MAF-7, H2O2) are almost the same as that of TMB-based chip alone (1) and the steady-state signals are approached in 9 days, indicating that HRP was well-sealed by the AptSTR outside the HRP@MAF-7 composites and would not produce false positive results. While upon addition of STR, the colorimeter signal remarkably increased and nearly remained constant in 9 days after the specific Apt-STR reaction (column (8)), suggesting that Apt@HRP/MAF-7 possessed satisfactory stability. Furthermore, only specific Apt-target recognition yielded an obvious increase of the signal, because no signal can be observed in column (9) in which the STR was replaced by KANA, indicating that there was no cross-reactivity in this platform. Clearly, all the encouraging results suggested that the Apt shell could firmly suppress the catalytic activity of HRP@ MAF-7 and could not be degraded/released from the particle surface along with the storage time. Additionally, it could be specifically triggered by specific Apt-target recognition. Therefore, sensitive and selective detection of the proposed colorimetric sensor is feasible. Experimental Conditions Optimization. Based on the well-preserved enzymatic activity and target response capability of Apt@HRP/MAF-7, we then conceive of a potential application of the fabricated Apt@HRP/MAF-7 composites in in-field detection of antibiotics, in which the TMB-MD was employed as the portable carrier for detection. Thus, the paper material used in the texture TMB-based chip was first evaluated. Compared with other paper materials, the filter paper (Whatman chromatography paper #1) could afford a better signal response (Figure S11) for its high hydrophilic, excellent retention of fine particles, and uniform pore size distribution. Besides, Apt-target incubation time, concentrations of TMB and H2O2, and catalytic reaction time, which might govern the reliability and sensitivity of this system, were also systematically optimized. As the results shown in Figure S12a, the colorimeter signal positively correlates with the
HRP, the catalytic kinetics of HRP@MAF-7 composites were then investigated. As the results shown in Figure S9, the initial rates versus TMB and H2O2 concentrations both follow typical Michaelis−Menten behavior in a certain range of substrate concentration. By using TMB as the substrate, the Km values (calculated by the Lineweaver−Burk plot, Table S3) of HRP@ MAF-7 and free HRP are approximate with each other. Nevertheless, when H2O2 served as the substrate, the Km value of HRP@MAF-7 was 4-fold lower than that of free HRP. It is known that the lower Km value reflects a higher affinity between enzymes and substrates.27 Meanwhile, owing to the enhanced affinity between substrate and encapsulated HRP, the Vmax values of HRP@MAF-7 composites are higher than that of free HRP. These results confirmed that the catalytic activity of HRP@MAF-7 composites was higher than that of free HRP, which would be contributed to the following reasons: (1) the guest molecule H2O2 (1.5 Å) is easy to diffuse through the pores of HRP@MAF-7 and the MAF-7 cavity confinement effect can build up local concentration of H2O2, accompanied by the synergy of the MAF-7 shell and incorporated enzyme,28 (2) the diameter and hydrophilic properties of pores in MAF-7 frame can achieve the catalytic selectivity that enable selective and fast diffusion of reaction reagents through the interior cavities, especially for reactions in liquid phase,29 (3) the structural stability and porosity of MAF7 crystals can reduce undesirable aggregation and atomic leaching of enzyme,30 and (4) the possible released Zn2+ ion seems to activate catalytic activity of HRP as observed in other enzymes (Figure S8c).31 Logically, the enhanced catalytic activity would benefit the sensitivity of HRP@MAF-7 based detections. Feasibility Investigation. Since the enzymatic activity of the encapsulated HRP was well preserved and even improved by MAF-7 frame, we then further investigated the shielding function, stability, and target response capability of the Apt shell out of the HRP@MAF-7 composites. As shown in Figures E
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Figure 5. (a) Calibration curve for STR detection (0, 0.005, 0.05, 0.1, 0.3, 0.5, 1, 3, and 6 ng/mL). (Inset shows photographs for visible detection of STR). (b) Selectivity of the detection of STR. The STR concentration is 2 ng/mL, and others are 1 μg/mL. (c) Calibration curve of KANA with concentrations of 0, 0.5, 1, 3, 5, 10, 30, 50, 70, 100, 200, and 300 pg/mL. (Inset shows photographs for visible detection of KANA). Experiment conditions: Apt@HRP/MAF-7: 2 mg/mL; TMB: 2.5 mM; H2O2: 10 mM; Target-Apt@HRP/MAF-7 incubation time: 20 min; and reaction time: 1 min. (Error bars, SD, n = 5).
Table 1. Comparison of Assay Results of the Developed Method and Commercial ELISA Kits for Detection of STR in Real Samples (n = 5)a this method sample raw milk
Xiangjiang river
ELISA kits
spiked (ng/mL)
found (ng/mL)
recovery ± RSD (%)
found (ng/mL)
recovery ± RSD (%)
0 1 3 5 0 1 3 5
0.95 2.97 4.91 1.03 3.08 5.07
95 ± 2.0 99 ± 2.3 98.2 ± 1.5 103 ± 1.6 102.7 ± 3.1 101.4 ± 1.8
3.13 5.08 3.12 5.11
104.3 ± 4.1 101.6 ± 2.4 104 ± 3.7 102.2 ± 2.8
“” means not found.
a
μg/mL). As the results illustrated in Figure 5b, a significant ΔC enhancement is obtained for the target STR (2 ng/mL), while negligible signal responses could be observed for other antibiotics, indicating that our detection possesses satisfactory specificity and selectivity. Besides, the reproducibility and stability of the proposed method were also evaluated. By testing STR samples with three concentrations (0.3, 0.5, and 1 ng/mL), the relative standard deviation (RSD) of the intraassay and inter-assay is calculated to be 1.49−4.45% (n = 5) and 4.43−5.78% (n = 5), respectively (Table S6), demonstrating the excellent reproducibility of the detection. Furthermore, the stability of this sensor was evaluated into two groups: one was stored in the room temperature and another was kept in 4 °C (in a refrigerator). As shown in Figure S13, the detection performances of two groups are all well preserved in 7 days but steadily decrease after 15 days for the room temperature group, and the refrigeration would extend the validity of this sensor to 55 days or even longer. These results indicated that this colorimetric sensor, especially for the one being refrigerated, exhibits considerable stability. Real Sample Analysis. To assess the practicability of this sensor, raw milk (obtained from a local market) and water from the Xiangjiang river with pretreatment spiked with different concentrations (1, 3, and 5 ng/mL) of STR were subjected for proposed method and ELISA kits, respectively. As the results shown in Table 1, favorable recoveries of ranged from 95 to 103% (n = 5) and accuracy were obtained for our method, and the developed method exhibited higher sensitivity than commercial STR ELISA kits. Therefore, the considerable performance of this portable platform would provide a
incubation time from 1 to 30 min and reaches a plateau after 20 min, indicating that the specific Apt-target reaction can be essentially completed in 20 min, while the signal also increases with the addition of TMB content and reaches a balance after 2.5 mM (Figure S12b), and for the concentration of H2O2, the ΔC signal increases with the increasing concentration of H2O2 but decreases upon the concentration over 10 mM (Figure S12c), which might be attributed to the inhibition of enzyme activity under H2O2 concentrations higher over 10 mM.32 Additionally, the enzyme-catalyzed reaction can reach equilibrium within 1 min (Figure S12d). Performance Evaluation. Under these optimized conditions, the analytical performance of this colorimetric sensor was then evaluated by adopting STR as the proof-of-concept target. As shown in Figure 5a, the signal response (ΔC value) of testing dot increases linearly with the increasing concentration of STR over the range of 0.005−6 ng/mL, and the LOD is estimated to be 0.51 pg/mL, which is significantly lower than that of existed point-of-care testing platforms (Table S4) and several commercial STR ELISA kits (e.g., R-Biopharm, 5 ppb; RESEARCH AREA, 2 ppb). Meanwhile, the favorable LOD is also superior to many reported laboratory methods (Table S5). In Table S5, although the LODs of some references are lower than that of our work, complicated probe preparation procedures, high-cost, or low mobility would prevent their application in antibiotics on-site monitoring. Remarkably, the proposed method is more portable, simple, and user-friendly than many traditional and commercial detection means. To verify the selectivity of this sensor in detecting STR, we challenged our detection by using other similar antibiotics (1 F
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Xiaoqing Chen − College of Chemistry and Chemical Engineering and Key Laboratory of Hunan Province for Water Environment and Agriculture Product Safety, Central South University, Changsha 410083, Hunan, China; orcid.org/ 0000-0002-8768-8965; Email: [email protected]
promising application for on-site sensitive detection of STR in food and environment safety monitoring. Universality and Expansibility of the Developed Platform. It is noticed that the antibiotics residues are multitudinous, and the universality of sensing platform is more demanding than ever before. Thus, we generalized the proposed detection method to other antibiotics by simple varying the Apt sequence. Herein, a common aminoglycoside KANA was selected as another target contamination.33 With a similar detection strategy, the ΔC intensity was recorded in the presence of different concentrations of KANA standard solution. As the results shown in Figure 5c, a good correlation between the concentration of the KANA and ΔC response can be obtained in relatively wide dynamic ranges (0.5−300 pg/ mL). The LOD was estimated as 0.34 pg/mL, which could be comparable to other reported methods (Tables S4 and S5). The considerable universality and expansibility of this platform would promise its great application in field-based detection of various antibiotics.
Authors
Lumin Wang − College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, Hunan, China Guangjuan Liu − College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, Hunan, China Yuxiang Ren − College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, Fujian, China Yinghui Feng − College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, Hunan, China Xinyi Zhao − College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, Hunan, China Yuqiu Zhu − College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, Hunan, China Miao Chen − College of Chemistry and Chemical Engineering and School of Life Science, Central South University, Changsha 410083, Hunan, China Fawei Zhu − College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, Hunan, China Complete contact information is available at: https://pubs.acs.org/10.1021/acs.analchem.0c03723
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CONCLUSIONS In summary, a sensitive colorimetric sensor has been favorably developed for on-site detection of antibiotics by employing Apt/HRP@MAF-7 composites as the target identification and signal transduction mode and the color change of TMB-based chip as the signal reporter via portable colorimeter readout. This assay can be easily operated in a tube. We demonstrated that the MAF-7 skeleton can not only offer excellent protection to HRP but also prominently enhance enzyme catalytic activity, which is beneficial for improving the sensitivity of detection. Particularly, we find that the Apt shell capped over the surface of MAF-7 can control the catalytic activity of HRP@MAF-7, guaranteeing the selectivity of this sensor by specific Apt-target recognition reaction. Moreover, the colorimeter is free from the influence of subjective interpretation of operator and external circumstances noise, endowing the developed sensor with excellent accuracy of detection. Significantly, this strategy can be easily generalized to on-site detection of other contaminations by simply altering the corresponding Apt sequence. On the basis of easy operation, high sensitivity, and versatility, the proposed platform paves a bright route for application of enzyme@MOFs composites and holds great potential for on-site detection of trace targets in environmental inspection, food safety/drug residue inspection, and health supervision..
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We gratefully acknowledge the financial support from the National Natural Science Foundation of China (21878339 & 21904141). This work was supported by The Key R&D Project in the industrial field funded by Hunan Provincial Science &Technology Department, China (2019GK2131).
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ASSOCIATED CONTENT
* Supporting Information sı
The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.analchem.0c03723. Reagents and materials, instruments, determination of enzyme concentration and encapsulation efficiency, activity assay, kinetic parameters, preparation of HRPon-MAF-7 and FHRP-on-MAF-7, SDS-PAGE analysis, pretreatment of real samples, detailed figures, tables, and references (PDF)
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AUTHOR INFORMATION
Corresponding Authors
Qi Liu − College of Chemistry and Chemical Engineering, Central South University, Changsha 410083, Hunan, China; Email: [email protected] G
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