Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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Hydrogel-Encapsulated Enzyme Facilitates Colorimetric Acute Toxicity Assessment of Heavy Metal Ions Yue Zhang,† Tingting Ren,†,‡ Hua Tian,† Binbin Jin,†,‡ and Junhui He†,* †
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Functional Nanomaterials Laboratory, Center for Micro/Nanomaterials and Technology and Key Laboratory of Photochemical Conversion and Optoelectronic Materials, Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, Zhongguancundonglu 29, Haidianqu, Beijing 100190, China ‡ University of Chinese Academy of Sciences, Beijing 100864, China S Supporting Information *
ABSTRACT: Conventional analysis of heavy metal ions in water requires highly skilled staff and sophisticated equipment. These limitations make conventional approaches difficult to perform analysis on-site without delay. Herein, we report a facile colorimetric sensing system developed for acute toxicity assessment of heavy metal ions. A bioactive enzyme, β-galactosidase, was used as sensing agent rather than bacteria or other higher organisms to improve selectivity and response time. The developed bioassay is capable of assessing the toxicity of heavy metal ions such as Hg(II), Cd(II), Pb(II), and Cu(II). The effects of enzyme concentration on the assessing performances (i.e., sensitivity and response time) of bioassay were explored and illustrated. Generally, low enzyme concentration facilitates sensitivity enhancement, achieving a 50% inhibiting concentration (IC50) of 0.76 μM (=152 ppb) Hg(II), and high enzyme concentration ensures quick response, enabling a response time down to 9 min. Moreover, the enzyme and substrate were respectively encapsulated by hydrogel to further simplify the assay procedure and enhance the stability of the enzyme. The hydrogel-encapsulated enzyme worked well even when heated up to 60 °C and retained ca. 90% activity after storage for 5 months. Moreover, the developed toxicity-assessing system is feasible for assessing toxicity of actual water samples. This assay approach is low cost and time effective and has no potential ethic issues. In addition, this work paves the way for the development of toxicity assessment kits for on-site analysis based on functional bioactive molecules. KEYWORDS: toxicity assessment, colorimetric assay, hydrogel encapsulation, heavy metal ions, visualized analysis
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INTRODUCTION Nowadays, heavy metal pollution as a global environmental issue has become a severe public health concern worldwide.1,2 Conventionally, analysis of heavy metal ions in water requires highly skilled staff and sophisticated equipment such as gas chromatography, atomic absorption spectroscopy, inductively coupled plasma atomic emission spectroscopy, and so on.3,4 Moreover, although these approaches can distinguish between varieties of heavy metal pollutants in water and provide relatively accurate concentration values, they are not able to reflect the impact of polluted water on active biospecies or living organisms. In addition, it is difficult to monitor all kinds of heavy metal pollutants in water simultaneously using the conventional approaches mentioned above. These limitations make conventional approaches difficult to perform analysis at the site of interest (on-site) without delay. The response of biological systems to pollutants within the environment is the conceptual basis for the development of acute toxicity assessment. The toxicity-assessing system not only allows an accurate biospecific determination of con© XXXX American Chemical Society
taminant concentrations but also provides information on the comprehensive impact of contaminants on biological active species and organisms from the perspective of life health. Acute toxicity assessment approaches based on microorganisms have attracted much attention.5,6 Reinhartz et al. reported toxicity assessment of heavy metal ions based on the stimulated biological production of β-galactosidase in Escherichia coli.7 They verified that the toxicity assessment system is sensitive to heavy metal ions (e.g., Ag(I) and Hg(II)), rather than to organic solvents (e.g., methanol, ethanol, and dimethyl sulfoxide). On the basis of this method, Environmental Biodetection Products Inc (EBPI) developed a kit named “ToxiChromoTest” for toxicity assessment of heavy metal ions. In addition, Bitton et al.8 developed another toxicity assessment kit “MetPLATE”, where chlorophenol red β-D-galactopyranoside (CPRG) is adopted as substrate instead of o-nitrophenylReceived: May 30, 2018 Accepted: July 19, 2018
A
DOI: 10.1021/acsami.8b08949 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 1. (A) Illustration of the assay principle for toxicity assessment. (B) Effects of various metal ions on β-galactosidase activity.
β-D-galactopyranoside. Recently, Zhi et al. reported acute biotoxicity assessment of heavy metal ions based on Bacillus subtilis, which could generate more β-galactosidase without the requirement of inducer compared to that by E. coli.9 It is noteworthy that all of the toxicity-assessing systems/kits mentioned above essentially rely on β-galactosidase although bacteria were adapted. All of the bioassays require a lysing procedure to release enzyme; otherwise, they suffer from slow response due to the diffusion process of heavy metal ions through the cell membrane. In addition, the sensitivity of the toxicity assessment systems based on bacteria is often unsatisfactory because one bacterium contains numerous proteins/enzymes, some of which may also interact with heavy metals. The successful development of biosensors is often hampered considerably by the instability of biological species (e.g., enzymes and cells). Encapsulation is one of the favorable strategies to offer a comfortable microspace for biological species and preserve their activity/viability under perturbations of extreme pH, temperature, and ionic strength changes.10−13 Current encapsulation materials include liposomal vesicle,14−16 polyelectrolyte,17 hydrogel,18−20 sol−gel,21,22 and so on. Among these encapsulation materials, hydrogel has been widely used due to its low cost, good biocompatibility, easy preparation, and no necessary use of organic solvent. DNA hydrogel was constructed and used to encapsulate enzymes for cascade reaction.23 This hydrogel exhibits enhanced multienzyme cascade reaction activity by trapping these enzymes in a microspace. In addition, well-defined hydrogel microarrays were created for entrapment of two enzymes without causing their deactivation.24 In this work, a new colorimetric bioassay for toxicity assessment of heavy metal ions was developed relying on βgalactosidase as sensing agent rather than whole bacterium. The effects of enzyme concentrations on the assessing performance of bioassay were explored systematically. Respective encapsulations of enzyme and substrate by hydrogel were performed to further simplify the assay procedure and enhance the stability of the enzyme.
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Shanghai Angyi Biological Technology Co. and Sigma-Aldrich (Shanghai) Trading Co., respectively. Copper chloride, cadmium chloride, zinc chloride, nickel nitrate, silver nitrate, magnesium chloride, ferric nitrate, cesium chloride, and lead nitrate were purchased from Beihua Fine Chemicals. The deionized water used in the experiments was obtained using a MilliQs Plus 185 purification system. MOPs buffer (pH = 7.2, 10 mM) was prepared and used in all assays. For actual river water sample test, water samples from the Qinghe river of Beijing, China, were obtained and filtered through a 0.22 μm membrane. Enzyme Solution-Based Toxicity Assessment. Solution-based assessing systems containing varied concentrations of enzymes (from 0.5 to 50 U/L) were adopted to evaluate the toxicity of samples containing various amounts of Hg(II). On account of the different concentrations of enzyme used, the time for enzymatic reactions was varied to achieve observable assay results. Specifically, for assessing systems contain 0.5, 1, 5, 10, 20, and 50 U/L enzyme, the enzymatic reactions proceeded for 17 h, 8 h, 2 h, 1 h, 30 min, and 20 min, respectively. The effects of various metal ions on β-galactosidase were investigated as well. The tested metal ions include Cr(VI), Ba(II), Fe(III), Mg(II), Ag(I), Ni(II), Zn(II), Cd(II), Pb(II), Hg(II), and Cu(II) with an identical concentration of 5 μM. For all of the assay systems, a substrate concentration of 0.5 mM was used unless otherwise stated. The absorption spectra were measured using a TU1901 spectrophotometer (Beijing Purkinje General Instrument Co.). The steady-state photoluminescence emission were measured on an F-4600 fluorescence spectrophotometer (Hitachi) by exciting the samples at 278 nm. Encapsulation of Enzyme and Substrate by Hydrogel. The enzyme and substrate hydrogels were prepared through a freeze−thaw approach. First, 10 g of PVA was mixed with 90 g of water. Then, the mixture was heated at 90 °C with stirring until clear solution was obtained. Next, 4 g of PVA solution (10 wt %) was separately mixed with 100 μL of enzyme solution (8000 U/L) and 320 μL of substrate solution (27 mM) to make mixtures. Following thorough mixing and degassing, the mixtures were separated into small aliquots (0.25 g each) and added into identical molds. After freezing in a fridge at −20 °C for 12 h and thawing at room temperature, the hydrogels were stored at 4 °C for later use. Mechanical and Hg(II) Sorption Properties of Hydrogels. The mechanical properties of the hydrogels were studied using a ferruled optical fiber-based nanoindenter (PIUMA, Optics 11, Amsterdam; The Netherlands) at room temperature. A probe with a tip diameter of 50 μm was used here. Measurements were performed at different positions of each hydrogel to receive an average value. For Hg(II) sorption kinetics test, 10 pieces of enzyme hydrogel were added to Hg(II) solution (7.5 μM = 1.5 ppm). Then, 4 mL of the solution was taken out at intervals, and its concentration of Hg(II) was measured by using inductively coupled plasma optical emission spectrometry (ICP-OES, Varian 710-OES). Enzyme Hydrogel-Based Toxicity Assessment. A piece of enzyme hydrogel and a piece of substrate hydrogel were added into aqueous solutions containing varied amounts of Hg(II). The
MATERIALS AND METHODS
Materials. Mercury standard solution (1000 μg/mL in 1 mol/L nitric acid) and poly(vinyl alcohol) (PVA, 98% hydrolyzed) were purchased from Shanghai Aladdin Biochemical Technology Co. 3-(NMorpholine) propanesulfonic acid (MOPS) was purchased from Nanjing Yuanbaofeng Pharmaceutical Technology Co. β-Galactosidase (>8 units/mg solid, from Aspergillus oryzae) and chlorophenol red-β-D-galactopyranoside (CPRG, 96%) were purchased from B
DOI: 10.1021/acsami.8b08949 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
Figure 2. Effect of enzyme concentration (0.5−50 U/L) on toxicity-assessing performance. (A−F) Absorption responses of Hg(II) titrations using assessing systems containing varied enzyme concentrations. Insets in (A) and (F) are photographs of the corresponding samples. (G) Calibration curves for the quantification of Hg(II) with different enzyme concentrations. (H) Dissociation constant K and the time required for absorbance of control samples to reach 0.5 (i.e., TimeA=0.5) as a function of enzyme concentration.
enzyme β-galactosidase is able to catalyze substrate CPRG producing colored chlorophenol red (absorbance at 575 nm), allowing absorption-based readout. However, in the presence of toxic heavy metal ions, the activity of the enzyme was inhibited and the colorimetric reaction was hindered. On the basis of this, the bioassay for toxicity assessment of heavy metal ions was developed. The effects of various metal ions on βgalactosidase were investigated and are shown in Figure 1B.
colorimetric reactions were monitored and photographed. Finally, the photographs were processed by Image J, and the light intensity values of samples were measured and plotted as a function of Hg(II) concentration.
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RESULTS AND DISCUSSION The enzyme (i.e., β-galactosidase) and substrate (i.e., CPRG) were used corporately in all bioassay systems for toxicity assessment in this work. As illustrated in Figure 1A, the C
DOI: 10.1021/acsami.8b08949 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
Research Article
ACS Applied Materials & Interfaces
A0 and AHg represent the absorbance values of the solution in the absence and presence of Hg(II), respectively.
The enzyme reserves its activity by at least 88% in the presence of Ba(II), Fe(III), Ni(II), Mg(II), and Zn(II). The enzyme activity was reduced by 21 and 27% by Ag(I) and Cd(II), respectively; Hg(II), Pb(II), and Cu(II) severely inhibited the enzyme activity by 53, 61, and 94%, respectively. This indicates that the developed system is capable of assessing toxic metal ions such as Cd(II), Hg(II), Pb(II), and Cu(II). It is also predictable that the system has the ability of assessing the total toxicity of water samples containing more than one toxic metal ion. The enzymatic colorimetric reaction rate was explored as a function of substrate concentration and enzyme concentration separately (see Figure S1). When subjected to fixed enzyme concentration, the reaction rate increases proportionally as the substrate concentration increases (Figure S1A,B); when subjected to fixed substrate concentration, the reaction rate increases proportionally as the enzyme concentration increases (Figure S1C,D). On account of this, the concentrations of enzyme and substrate used in this study were in the range of 0.5−200 U/L and 1−5 mM, respectively, to ensure that the colorimetric reactions fall within the linear response range, facilitating quantitative assessment of toxic heavy metal ions. As a model toxic heavy metal ion, the destructive effect of Hg(II) on β-galactosidase was systematically examined. As can be seen from Figure 2A, after the addition of Hg, the activity of the enzyme was inhibited gradually. Meanwhile, the effect of enzyme concentration on the toxicity-assessing performance was evaluated by using varied concentrations (0.5−50 U/L) of β-galactosidase and a fixed concentration of CPRG (5 mM) to quantitate Hg(II). To produce distinguishable signals, the reaction time was tailored according to the enzyme concentration of each system. As the enzyme concentration increases, the reaction time decreases and vice versa. Figure 2A−F shows the absorption responses of Hg(II) titrations using assessing systems containing varied enzyme concentrations. In these systems, the catalytic activity of the enzyme is proportional to its effective concentration, and the absorbance, A, at 575 nm was used to indicate its catalytic activity A = a + b × [E]
A 0 − AHg [E·Hg] [Hg]free h = = [E]total A0 − a [Hg]free h + K h AHg = A 0 − (A 0 − a) ×
AHg = A 0 − (A 0 − a) ×
(2)
According to eq 1 A0 − a b
[E·Hg] = [E]total − [E]free = =
(3)
AHg − a A0 − a − b b
A 0 − AHg b
[Hg]free h + K h
(6)
[Hg]total h [Hg]total h + K h
(7)
All of the absorbance values at 575 nm corresponding to Hg(II) concentrations were extracted from Figure 2A−F and normalized to 1 to facilitate data fit and comparison. Figure 2G demonstrates the obtained curves fitted by the Hill equation, with each data point representing the average value of two parallel experiments. The fitting parameters (i.e., h and K) are provided in Table S1. For all assay systems, the Hill coefficients, h, obtained were found to be all greater than 1, suggesting that the self-assembly interaction between βgalactosidase and Hg(II) is positively cooperative, i.e., once one Hg(II) is bound to an enzyme, the affinity of the enzyme for a second Hg(II) is increased. According to the definition of dissociation constant, assuming one enzyme only chelates one Hg(II), K represents the concentration of free Hg(II) (i.e., [Hg]free) at which half of the total enzyme molecules are associated with Hg(II). Assuming that [Hg]free ≈ [Hg]total and the enzyme−Hg complex has no catalytic activity, K indicates the concentration of Hg(II) at which half of the total enzyme molecules lose their activities, i.e., 50% inhibiting concentration (IC50). As shown in Figure 2H, as the enzyme concentration increases, the dissociation constant, K, increases at first, but achieves a maximum value at 20 U/L. It can be interpreted that the sensitivity of the assessing system decreases as the enzyme concentration used increases in the range of 0.5−20 U/L. That means, low enzyme concentration facilitates sensitivity enhancement, achieving an IC50 down to 0.76 μM.26,27 Nevertheless, as the enzyme concentration increases, the times required for the absorbance of control samples (without heavy metals) to reach 0.5 decrease significantly. It implies that high enzyme concentration ensures a high colorimetric reaction rate, i.e., quick response enabling a response time down to 9 min. In short, low enzyme concentration facilitates high sensitivity, whereas high enzyme concentration ensures quick response generally. This indicates a compromise between the sensitivity and the response time. In practical applications, an analyst can choose the right system according to the actual situation. The sensing behaviors of the bioassay for Pb(II), Cd(II), and Cu(II) titrations were also examined and are demonstrated in Figure S2 and Table S2. In addition, the sensing performance of the developed toxicity assessment assay is compared to that in previously reported works and is shown in Table S3. It indicates that the sensitivity of the asdeveloped toxicity assessment assay based on β-galactosidase is better than those assays using E. coli as the sensing agent.9,26,28−33
In eq 1, [E] represents the concentration of active enzyme. When Hg(II) was added into the system, Hg(II) coordinated with the enzyme, hampering its catalytic activity. [E·Hg] and [Hg] represent the concentrations of enzyme−Hg complex and Hg(II), respectively. The general form of the Hill equation is as follows, where h and K represent the Hill coefficient and the dissociation constant, respectively25
[E]total =
[Hg]free h
The condition under which [Hg]free ≈ [Hg]total is [Hg]free ≫ [E·Hg], which usually requires [Hg]total ≫ [E]total. In this study, the above conditions are satisfied, so the Hill equation utilized here to fit the titration data is defined as
(1)
[E·Hg] [Hg]free h = [E]total [Hg]free h + K h
(5)
(4) D
DOI: 10.1021/acsami.8b08949 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
Figure 3. (A) Fluorescence of the enzyme incubated with varied concentrations of Hg(II). (B) Relationship between the fluorescence ratio and the Hg(II) concentration.
Figure 4. (A) Mechanical properties of the enzyme and substrate hydrogels. (B) Hg(II) sorption capability of the enzyme hydrogel. (C) Visualized and quantitative hydrogel-based toxicity assessment of the samples with varied Hg(II) concentrations. (D) Illustration of the hydrogel-based toxicity assessment manual and the color evolution of the samples with varied Hg(II) concentrations.
Fluorescent spectrometry is an effective method to study the molecular conformations of proteins because the fluorescent parameters reflect the conformation and structure of molecules from all angles. By inferring the conformational change, the relationship between structure and function of protein could be illuminated. Tryptophan, tyrosine, and phenylalanine in protein molecule endow the protein with endogenous fluorescence, which could be used to characterize protein. Fluorescence quenching requires molecular contact between the fluorophore and quencher. This contact can be caused by diffusive encounters (i.e., dynamic quenching) or nonfluorescent complex formation (i.e., static quenching). In this study, the quenching effect of Hg(II) on β-galactosidase was investigated. As shown in Figure 3, Hg(II) is an effective
quencher of the enzyme. As the concentration of Hg(II) increases, the fluorescence of the enzyme decreases gradually. Because the enzyme activity was inhibited by Hg(II) implying the formation of Hg−enzyme chelate complex, the fluorescence quenching process is assumed to be static quenching. The photoluminescence quenching was analyzed using the equation F0/F = 1 + Ks[Q]. Here, F0 and F are the fluorescence intensities in the absence and presence of Hg(II), respectively; Ks is the static quenching constant; and [Q] is the concentration of Hg(II). As can be seen from Figure 3B, when the concentration of Hg(II) is lower than 20 μM, the relationship between F0/F and Hg(II) concentrations is proportional. If the concentration of Hg(II) increases up to 35 μM, the photoluminescence data plot deviates from E
DOI: 10.1021/acsami.8b08949 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
Figure 5. Enzyme relative activities under (A) different temperatures and (B) different storage times. Insets in (A) are the corresponding photos of colorimetric assays.
concentration up to 50 μM hardly changed, indicating that the enzyme activity was almost completely suppressed. To achieve quantitative analysis, the photograph was processed by Image J software, and the gray-scale light intensity of each sample was measured and plotted. Figure 4D demonstrates the cartoon of the toxicity assessment system corresponding to colorimetric reaction evolution. This cartoon provides clues for design of operation manual and color-comparison card should the hydrogel-encapsulated enzyme system be developed as a toxicity assessment kit or applied for on-site toxicity assessment. In practical applications, the thermal stability and long-term validity of enzymes are big challenges. In this work, the high temperature tolerance of the enzyme in solution and hydrogel systems was examined and compared. The enzyme solution and hydrogel were incubated in various temperatures for half an hour at first. After colorimetric reaction, the absorbance of the supernatants of the enzymatic reaction resultants was recorded for each system. The enzyme relative activity was calculated using the activity of the enzyme at 25 °C as reference. As can be seen from Figure 5A, for the enzyme solution, the enzymatic activity first decreases slowly (from 25 to 50 °C) and then drops sharply (from 50 to 70 °C) as the incubation temperature increases. For the enzyme hydrogel, the enzymatic activity increases slowly as the incubation temperature increases from 25 to 55 °C. Basically, the hydrogel melts as temperature increases, leading to the leakage of the enzyme to the supernatant and resulting in the apparent rises of the enzymatic activity in the beginning. As the incubation temperature further increases from 55 to 70 °C, the hydrogel scaffold loses protection ability, resulting in that the activity of the enzyme hydrogel shows a similar sharp decrease to that of enzyme solution. Neither the enzyme in solution nor that in hydrogel functioned properly at 70 °C, with only 3.5 and 7.1% activity remaining, respectively. It should be noted that at 55 °C, the enzymatic activity of the enzyme hydrogel was ca. 40% higher than that of enzyme solution. In addition, the activities of the enzyme solution and enzyme hydrogel freshly prepared and after 5 months of storage were also examined and compared. Figure 5B shows that after storage for 5 months at 4 °C, the enzyme hydrogel maintained good performance with ca. 90% of its original activity left, whereas the enzyme solution lost ca. 60% activity. Therefore, it is concluded that the hydrogel system is able to provide protection for encapsulated enzymes in its framework. Enzyme encapsulation by hydrogel demonstrates an excellent alter-
linearity. The possible reason is that the secondary structure of the enzyme is disrupted by Hg(II) gradually. A previous study has indicated that Hg(II) forms mercaptides with SH and S−S groups of β-galactosidase.34 To simplify the analysis operation and facilitate the development of analytical kits, the enzyme and substrate were encapsulated by hydrogel separately. PVA was used here as the cross-linking matrix of hydrogel due to its good biocompatibility and stability. Freeze−thaw approach was employed to execute hydrogel encapsulation through physical cross-linking, rather than chemical covalent cross-linking approach, which may hamper the enzyme activity. As indicated in Figure 4A, a box-and-whisker plot shows the mechanical properties of the hydrogels, measured as effective Young’s modulus using a nanoindenter (n = 5). The box-andwhisker plot indicates the median (middle lime), mean (small square), 25 and 75% of values (box lower and upper bounds, respectively), and minimum and maximum values (whiskers). The enzyme and substrate hydrogels exhibited similar mechanical properties with effective Young’s moduli calculated to be 3.6 and 3.1 kPa, respectively. As shown in Figure 4B, the introduction of enzyme hydrogel leads to a rapid decrease in the Hg(II) concentration, with ca. 40% Hg(II) adsorbed by hydrogel within half an hour. Subsequently, the Hg(II) concentration leveled off at relatively stable values within 1 h. With a further prolonged adsorption time, the Hg(II) concentration increased slightly. The reason for that may be because the slight swelling of the hydrogel framework facilitated the release of enzyme and noncross-linked PVA molecules, which contributed to the adsorption of Hg(II), which thus stayed in the solution for ICP measurements. Overall, enzyme hydrogel is able to absorb and concentrate Hg(II), enhancing the local concentration of Hg(II), and therefore demonstrates potentials in improving sensing performance. To test the toxicity-assessing performance of as-prepared hydrogels, a piece of enzyme hydrogel and a piece of substrate hydrogel were added into aqueous samples containing varied amounts of Hg(II). Figure 4C shows the experimental results of the toxicity-assessing assays employing the hydrogelencapsulated enzyme and substrate. As the concentration of Hg(II) increases, the color change of the system becomes insignificant, implying that the enzyme loses its activity gradually. After 2 h of enzymatic reaction, the color differences between each sample are clear enough for visual distinction. It is noticeable that the color of the system with Hg(II) F
DOI: 10.1021/acsami.8b08949 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
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ACS Applied Materials & Interfaces
Figure 6. (A) Absorption responses of the bioassays based on enzyme hydrogel for toxicity assessment of actual water samples. (B) Actual concentrations of each metal ion in water samples measured by ICP. Note that “River water1” and “River water2” denote river water spiked with varied amounts of Pb(II), Cu(II), Cd(II), and Hg(II). Inset shows the photographs of the corresponding samples.
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native to maintain enzyme activities in harsh environment or for long-term storage. This facilitates the development of practical and commercial assays that require the reagents to have a long shelf life. To test the feasibility of the as-developed bioassay for the toxicity evaluation of real water samples, a standard addition method was applied to test water samples from the Qinghe river of Beijing, China. The river water samples were spiked with four kinds of toxic heavy metal ions, i.e., Pb(II), Cu(II), Cd(II), and Hg(II). In addition, the actual concentrations of each metal ion in water samples were measured by ICP and are shown in Figure 6B. As indicated in Figure 6A, the river water inhibited the enzyme activity by 30%. Additional spiked toxic heavy metal ions further inhibited the enzyme activity. These results imply that the approach developed here has the potential to assess the actual toxicity of water bodies. Upon further optimization, it is expected that heavy metal ion toxicity assessment kits could be developed for on-site analysis.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b08949. Dependence of enzymatic reaction rate on substrate and enzyme concentrations; absorption responses of Pb(II), Cd(II), and Cu(II) titrations; fitting parameters of calibration curves; comparison of the sensing performances of the developed toxicity assessment assay with previously reported works (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Fax: +86 10 82543535. ORCID
Junhui He: 0000-0002-3309-9049 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was financially supported by the National Key Research and Development Program of China (2017YFA0207102), the Innovative Talents Cultivation Project of Technical Institute of Physics and Chemistry, the Chinese Academy of Sciences (2014-Z), the National Natural Science Foundation of China (21571182), and the Science and Technology Commission of Beijing Municipality (Z151100003315018).
CONCLUSIONS
A facile colorimetric assay was developed for acute toxicity assessment of heavy metal ions. β-Galactosidase was adapted as a probe rather than bacteria or higher organisms. This endows the bioassay with not only high sensitivity and selectivity, but also quick responsivity. This bioassay is capable of assessing toxicities of heavy metal ions such as Hg(II), Cu(II), Pb(II), and Cd(II). The enzyme concentration-dependent assessing performances were demonstrated systematically. Generally, low enzyme concentration facilitates sensitivity enhancement, achieving an IC50 of 0.76 μM, and high enzyme concentration ensures quick response, enabling a response time down to 9 min. Through encapsulation of the enzyme and substrate by hydrogel, not only the operation procedures of the assay were simplified but also the enzyme stability was enhanced. Encapsulated enzyme by hydrogel worked well even heated up to 60 °C and maintained ca. 90% activity after storage for 5 months. This assay approach is low cost and time effective and has no potential ethic issues. Moreover, this assay approach could be expended for toxicity evaluation of other hazardous substances by selecting specific biomolecules. This work paves the way for the development of toxicity assessment kits for onsite analysis based on functional bioactive molecules.
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DOI: 10.1021/acsami.8b08949 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX
