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Photoelectrochemical biosensor for histone acetyltransferase detection based on ZnO quantum dots inhibited photoactivity of BiOI nanoflower
Sensors & Actuators: B. Chemical 307 (2020) 127633
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Photoelectrochemical biosensor for histone acetyltransferase detection based on ZnO quantum dots inhibited photoactivity of BiOI nanoflower
T
Yan Chen, Yunlei Zhou*, Huanshun Yin, Fei Li, Hui Li, Runze Guo, Yihao Han, Shiyun Ai College of Chemistry and Material Science, Shandong Agricultural University, 271018, Taian, Shandong, People’s Republic of China
A R T I C LE I N FO
A B S T R A C T
Keywords: Histone acetylation Photoelectrochemical biosensor BiOI nanoflower Antibiotic ZnO quantum dots
Histone acetylation is a very important post-transcriptional modification, which is catalyzed by histone acetyltransferase with acetyl group donor of acetyl coenzyme A (Ac-CoA). Abnormal histone acetylation is associated with many diseases, so it is important to develop a sensitive and efficient method for detecting histone acetyltransferase (HAT). In this paper, a simple and sensitive photoelectrochemical biosensor was established for indirect detection of HAT using the acetylation reaction by-product of coenzyme A (CoA) as target molecule. To construct the biosensor, BiOI nanoflower was employed as photoactive material, Fe3O4-NH2 and 3-maleimidopropionic acid were used as “bridge” reagents for immobilizing CoA, and ZnO quantum dots were adopted as photocurrent inhibitor. Under optimal experimental conditions, the photocurrent decreased with changing HAT concentration from 0.01 to 500 nM. The linear relationship between the photocurrent and the logarithm value of HAT concentration can be constructed with this concentration range and the detection limit was 3 pM (S/N = 3). The sensor has excellent detection specificity, good stability and reproducibility. Based on the current situation of serious antibiotic contamination, the effect of antibiotics on HAT activity was investigated using this photoelectrochemical biosensor.
1. Introduction Histone acetylation is an important covalent post-translational epigenetic modification, which plays crucial role in transcription, DNA replication, DNA damage/repair, and histone deposition [1]. Histone acetylation is catalyzed by histone acetyltransferase (HAT), which transfers acetyl group from acetyl donor of acetyl coenzyme A (Ac-CoA) to lysine residue of histone [2]. In this catalysis process, the donor of Ac-CoA is then changed to coenzyme A (CoA). It has been reported that histone acetylation has a significant effect on neurological disorders and is associated with metabolic syndrome, chronic inflammation, HIV infection, as well as cancer [3]. To further understand the biological functions of histone acetylation, the possible indication effect on diseases and relevant drug screening, the determination of histone acetylation and HAT with simple, sensitive and selective method is necessary. Traditional technique for HAT detection mainly relied on radioisotope labeling [4], which is limited due to the high risk to human health under radioactive exposure. Thus, various alternative techniques have been developed, including electrochemiluminescence [5–7], fluorescence [8–10], electrochemistry [11], time-resolved luminescence and [12] colorimetry [13]. Some of them depend on antibody and ⁎
enzyme; some of them depend on the resistance of peptide to protease hydrolysis and the charge properties with and without acetylation; some of them depend on the by-product (CoA) of acetylation process. Though these methods achieve the sensitive and selective detection of HAT, new method is also highly desirable with merits of simple operation, rapid response, inexpensive instrument, high sensitivity and specificity. Photoelectrochemical technique, which combines the advantages of optical method and electrochemical method, attracts wide attention because of its low cost, simple operation, low background signal and high sensitivity [14]. Up to now, various fields including DNA [15], microRNA [16], protein [17], small biomolecules [18], metal ions [19] have been infiltrated by photoelectrochemical sensors, which strongly demonstrates the widely applicability of photoelectrochemical detection platform. However, the application of photoelectrochemical biosensor for HAT detection is missed. As one of the important parameters for PEC biosensor, the origin of the photoelectrochemical signal may greatly influence the detection sensitivity, which mainly depends on the photoactive material. In recent years, various semiconductor materials including ZnO [20,21], TiO2 [22], C3N4 [23], BiOI [24] have been applied to the construction of PEC sensors. Among them, BiOI has attracted widespread concern due to its narrow band gap (1.8 eV) [25]
Corresponding author. E-mail address: [email protected] (Y. Zhou).
https://doi.org/10.1016/j.snb.2019.127633 Received 5 November 2019; Received in revised form 23 December 2019; Accepted 24 December 2019 Available online 28 December 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
Sensors & Actuators: B. Chemical 307 (2020) 127633
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irradiation source. To eliminate the ultraviolet light, an optical filter was equipped on the Xe lamp source. Three-electrode system was employed, where ITO or modified ITO electrode was used as working electrode, saturated calomel electrode was used as reference electrode, and platinum electrode was used as counter electrode. The applied potential is -0.4 V.
and strong visible light absorption ability, showing excellent optical and electrical properties [26]. For examples, Zhou et al. developed a sensitive photoelectrochemical biosensor for DNA methyltransferase activity assay using BiOI as a photoactive material [27]; Gong et al. successfully implemented biofunctional crossed BiOI flake arrays to achieve highly sensitive photoelectric biosensor for organophosphate pesticide [18]. In view of the excellent feature of photoactivity, BiOI is promising in the fabrication of photoelectrochemical biosensor with superior performances. Recently, ″signal-off″ model photoelectrochemical biosensor has attracted more attentions. As a kind of photoactive material, quantum dots (QDs) received widely applications in photoelectrochemical biosensor [28,29]. In view of the QDs has many unique physical effects, such as good biocompatibility, strong stability, leading to its great applications in photoelectric materials, biofluorescence labeling and so on [30]. Among various QDs, ZnO QDs attract our attentions due to the reaction activity of Zn2+ with phosphate group and the inhibition activity of ZnO to other photoactive materials [31,32]. As a kind of byproduct of histone acetylation, CoA has phosphate group, which can facilitate the capture of ZnO QDs to quench the photoelectrochemical signal. In this work, a simple, sensitive and selective photoelectrochemical method was developed for indirect HAT detection. As the by-product of the acetylation reaction catalyzed by HAT, CoA was employed as detection target molecule. BiOI nanoflowers were used as photoactive material and ZnO-QDs were used as photoelectrochemical signal inhibitor. To evaluate the applicability of the developed method, the effect of antibiotics on HAT activity was investigated, which may provide new mechanism on ecotoxicological effect antibiotics, offer new biomarker and new technique on ecotoxicological effect evaluation.
2.2. Synthesis of BiOI nanoflower BiOI nanoflower was prepared according to previous report [33]. In general, 1.94 g of Bi(NO3)3 was dissolved in 70 mL of ethylene glycol, followed by the addition of 0.664 g of KI. The ratio of n(Bi(NO3)3 and n (KI) was 1:1. After completely dissolved under magnetic stirring, the solution was transferred into a Teflon-lined autoclave and heated at 160 °C for 12 h. After the autoclave was cooled down naturally to room temperature, the precipitate was separated by centrifuging at 4000 rom for 10 min, which was then washed with water and ethanol for three times. Finally, the prepared BiOI was dried at 60 °C. 2.3. Preparation of Fe3O4-NH2 Fe3O4 was prepared based on previous paper by slightly modifying the previous paper [34]. In summary, 4.0 g FeCl2·4H2O and 9.5 g FeCl3·6H2O were dissolved in a three-necked flask containing 110 mL of deionized water and filled with N2 to eliminate the effect of O2. Then, 20 mL of 25 % ammonia water was added into the reaction system, which was then heated to 90 °C for 30 min. The precipitate was collected by the separation of magnet with washing with water and ethanol for several times. Finally, the prepared Fe3O4 was dried at 60 °C. To prepare Fe3O4-NH2, the obtained Fe3O4 and 200 mL ethanol were added into a three-necked flask under N2 atmosphere. The dispersion was heated to 75 °C. Then, 5 mL of 95 % APTES was further poured into the reaction system. After magnetic stirring for 2 h, the precipitate was separated by a magnet, which was then washed with water and ethanol for several times. Finally, the prepared Fe3O4-NH2 was dried at 60 °C.
2. Experimental section 2.1. Reagents and apparatus Horseradish peroxidase (HRP), alkaline phosphatase (ALP), bovine serum albumin (BSA) were obtained from Sigma-Aldrich (USA). Protein kinase A (PKA) was provided by NEB (USA). Histone acetyltransferase p300 (HAT) was supplied by Enzo. Biochem. (USA). The substrate peptide with the specific sequence of RGKGGKGLGKGGAKA was synthesized by Sangon (China). Bi(NO3)3, KI, FeCl2·4H2O, FeCl3·6H2O, NaH2PO4, Na2HPO4, NH3·H2O, Zn(CH3COO)2▪2H2O, CH3OH, disodium ethylenediaminetetraacetic acid (EDTA), (3-Aminopropyl)triethoxysilane (APTES), Ac-CoA (Enzymatic ≥ 83 %), CoA, tris (2-carboxyethyl)phosphine (TCEP), tris(hydroxymethyl) aminomethane (Tris), tobramycin, amoxicillin and tetracycline were purchased from Aladdin (China). A 96-well detachable ELISA plate was supplied by Corning Costa (USA). ITO conductive glass was purchased from Kaivo Electronic Components Co., Ltd. (Zhuhai, China, ITO coating 180 ± 25 nautical miles, sheet resistance < 15 Ω/cm2). Other unspecified reagents were of analytical grade and were not further purified. The buffer solutions used in the experiment were as follows. Washing buffer: 10 mM Tris−HCl, 50 mM NaCl (pH 7.4). HAT reaction buffer: 10 mM Tris−HCl, 50 mM NaCl and 10 mM MgCl2 (pH 7.4). Photoelectrochemical detection buffer: 0.1 M PBS containing 0.1 M AA (pH = 7.4). All solutions were prepared using distilled deionized water produced by DEPC. Scanning electron microscopy (SEM) images were recorded by Hitachi S-4800 SEM (Japan). JEM-2100 microscope (Japan) was used to measure transmission electron microscopy (TEM) images. Fourier transform infrared spectroscopy (FT-IR) was determined by Thermo Nicolet-380 IR spectrophotometer (USA) in the range of 4000 – 400 cm−1. The crystal structure of the material was analyzed by X-ray diffraction (XRD, Cu Kα, Rigaku, D/max 2500, Japan) at room temperature. Photoelectrochemical measurement was performed on a CHI832A electrochemical station with a 500 W Xe lamp as the
2.4. Synthesis of ZnO QDs ZnO QDs were prepared according to previous work using ZnO nanospheres as precursor [32,35]. Briefly, 0.1 M zinc acetate dihydrate was dissolved into 50 mL of methanol under magnetic stirring. After that, 25 mL of 0.5 M NaOH solution in methanol was further added into the above solution. After magnetic stirring for 30 min, the solution was then poured into a Teflon-lined stainless-steel autoclave and heated at 150 °C for 6 h. After naturally cooled to room temperature, the sediment was collected by centrifugation, which was washed with water and ethanol for several times. The prepared ZnO nanospheres was dried at 60 °C. 7.5 mg ZnO nanospheres was dispersed into 15 mL deionized water with the aid of ultrasonication for 1 h. Then, 1 mL of 0.1 M ascorbic acid aqueous solution was further added into the dispersion, which was transferred to a Teflon-line stainless steel autoclave and heated at 200 °C for 2 h. After cooling to room temperature naturally, it was centrifuged at 4000 rpm for 10 min to remove impurities, and the dispersion was collected. Finally, the dispersion was poured into a dialysis membrane for 12 h of dialysis. 2.5. Acetylation reaction Firstly, 30 μL 2 × HAT reaction buffer containing different concentration of HAT (p300) and 20 μM Ac-CoA were added into a centrifugal tube. Then, 10 μL of 6 μM substrate peptide was further added into the centrifugal tube. The centrifugal tube was oscillated for two hours in humid environment at 37 °C. Afterwards, the solution was 2
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catalysis process, acetyl group of Ac-CoA can be transferred to the lysine residue of substance peptide, and CoA can be produced as a byproduct. CoA can be easily recognized and captured due to the thiol group and phosphate group in its structure. Thus, CoA was employed as target molecule to achieve the indirect detection of HAT. Fig. 1B illustrates the fabrication of photoelectrochemical biosensor and HAT detection. Firstly, BiOI was immobilized on the bare ITO electrode surface as photoactive material. Then, Fe3O4-NH2 was modified to provide –NH2 on the electrode surface. Afterwards, based on the covalent reaction between –NH2 and −COOH [36], 3-maleimidopropionic acid (MIPA) was further modified on the electrode surface, which could easily reacted with thiol group [37,38] Thus, CoA was then captured and its phosphate group was away from the electrode surface. Finally, based on the specific interaction between ZnO and phosphate group [39], ZnO QDs were modified on the electrode surface. According to the change of the photocurrent, HAT can be detected.
transferred to a 96-well removable ELISA plate and incubated for 30 min, after which the supernatant was transferred to a centrifugal tube and stored at 4 °C before use (the resulting solution was designated as Target Solution). 2.6. Fabrication of the biosensor The ITO conductive glass (1 × 5 cm) was pretreated with the aid of ultrasonication with acetone and ethanol/NaOH solution (v/v, 1:1), respectively. Then, the electrode was rinsed with double distilled water and dried naturally at room temperature. After that, 40 μL of 4 mg/mL BiOI dispersion was dropped onto the electrode surface, and dried under the irradiation of an infrared lamp (The electrode was named BiOI/ITO). Subsequently, 40 μL of 0.5 mg/mL Fe3O4-NH2 dispersion was further casted onto the electrode surface and dried under the irradiation of infrared lamp (The obtained electrode was named Fe3O4/ BiOI/ITO). After that, 40 μL of 2 mg/mL 3-maleimidopropionic acid (MIPA) was dropped onto Fe3O4/BiOI/ITO electrode surface and incubated for 120 min in a humidified environment at 37 °C, where the 3maleimide propionic acid was activated by 1 μM EDC and 1 μM NHS before use. The prepared electrode was denoted as MIPA/Fe3O4/BiOI/ ITO. Afterwards, the electrode was incubated with 40 μL Target Solution (described in section 2.5) for 120 min in a humid cell at 37 °C. The obtained electrode was labeled as CoA/MIPA/Fe3O4/BiOI/ITO. Finally, the electrode was incubated with 40 μL ZnO QDs under humid conditions for 120 min. The prepared electrode was named as ZnO/ CoA/MIPA/Fe3O4/BiOI/ITO. After each modification process, the electrode was rinsed three times with washing buffer.
3.2. Characterization of nanomaterials The morphology and elemental distribution of BiOI were analyzed by SEM and mapping, respectively. As depicted in Fig. 1A, the prepared BiOI was a flower-like spherical structure with an average diameter of about 2 μm, which was stacked by a plurality of staggered sheet structures with thickness of 10−20 nm. The distribution of elements in the BiOI was further determined by EDS. The results indicate that the elements of the prepared material are evenly distributed. The morphology of Fe3O4-NH2 was investigated by TEM, which showed a relatively uniform distribution, but slightly agglomerated, and the particle size was between 12 and 16 nm (Fig. 1C). Fig. 1D presented the TEM and HRTEM images of ZnO QDs. The lattice spacing of 0.26 nm corresponds exactly to the (002) crystal plane of ZnO (JCPDS No. 36–1451) [11], indicating the successful preparation of ZnO QDs. XRD was employed to further explore the crystal configuration of synthetic BiOI. As illustrated in Fig. 2A, the crystal structure of BiOI corresponds exactly to JCPDS card No. 10-0445, indicating the successful preparation of BiOI with a standard tetragonal phase [40]. The XRD patterns of Fe3O4 and Fe3O4-NH2 were depicted in Fig. 2B. The positions of the diffraction peaks are basically consistent before and after amino modification, indicating that the amino modification does not change the crystal form of Fe3O4, but the diffraction peak was slightly reduced, which could be attributed to the reduction of the crystallinity of Fe3O4 by amino modification [3]. In order to more accurately understand the successful modification of the amino group, the FT-IR was measured (Fig. 2C). The absorption peak at 588 cm−1 was derived from the stretching vibration of Fe-O, demonstrating the successful synthesis of the Fe3O4 nanospheres (black line). After treatment with APTES, the absorption peaks of 3135 and 1105 cm−1 was assigned to the stretching vibration of NeH bond and stretch vibration of Si-O bond, respectively. These results prove the successful amino-
2.7. Effect of antibiotic on HAT activity assay The effects of antibiotics on HAT activity were assessed. To perform it, different concentrations of antibiotics (tobramycin and amoxicillin) were added to the acetylation reaction system as described in Section 2.5, followed by the construction of the biosensor as described in Section 2.6. The results were evaluated by comparing the photocurrent change ratio (PCR (%) = (I1 – I2)/I2) of the biosensor with and without antibiotic, where I1 represented the photocurrent of ZnO/CoA/MIPA/ Fe3O4/BiOI/ITO with agents, and I2 was the photocurrent of ZnO/CoA/ MIPA/Fe3O4/BiOI/ITO without agents. 3. Results and discussion 3.1. Detection strategy In this paper, a photoelectrochemical biosensor was developed for HAT detection based on the by-product of CoA in the acetylation process of peptide catalyzed by HAT. In this detection strategy, the indirect HAT detection can be achieved using CoA as detection target. As shown in Scheme 1A, the amount of CoA is related to HAT activity. In the
Scheme 1. (A) Mechanism of the acetylation of short peptide catalyzed by HAT. (B) Schematic diagram of the construction of PEC biosensor for detecting HAT. 3
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Fig. 1. SEM image (A) and mapping image (B) of BiOI. Inset: SEM image of partially enlarged BiOI. TEM image of Fe3O4-NH2 (C). TEM and HRTEM (inset) image of ZnO Quantum Dots (D).
electrodes were experimentally determined and contrasted. As clearly shown in Fig. 3, a strong photocurrent was produced when BiOI was attached onto the bare ITO electrode surface (curve a), indicating that the excellent photoactivity of BiOI [24,41]. After the modification of Fe3O4-NH2 to the BiOI/ITO surface (curve b), the photocurrent intensity significantly increased, which may be attributed not only to the good electrical conductivity of Fe3O4, but also due to the heterojunction formed by the synergistic effect of Fe3O4 and BiOI, which increased the absorption of visible light, facilitating the generation, transfer and the separation of photo-generated electrons and holes [40,42]. Afterwards,
functionalization of Fe3O4 (red line) [3]. FT-IR was also employed to characterize ZnO QDs. As displayed in Fig. 2D, the peak at 470 cm−1 belonged to the Zn-O bond, which was the characteristic peak of ZnO [4]. Integrating the results in Figs. 1D and 2 D, ZnO QDS had been successful prepared.
3.3. Detection feasibility assay In order to verify the practicability of the prepared photoelectrochemical biosensor, the photocurrent responses of different
Fig. 2. XRD patterns of BiOI (A), Fe3O4 and Fe3O4-NH2 (B). (C) FT-IR image of Fe3O4 (black line) and Fe3O4-NH2 (red line). (D) FT-IR image of ZnO QDs (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). 4
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3.4. Optimization of experimental conditions In order to maximize the performance of the biosensor, several influencing factors were optimized, including BiOI concentration, MIPA incubation time, CoA reaction time and ZnO quantum dots immobilization time. As shown in Fig. 4A, the photocurrent intensity enhanced with changing BiOI concentration from 0.5 to 4 mg/mL. With increasing BiOI concentration, more photogenerated electrons produced, which led to the increased photocurrent. However, when further increasing BiOI concentration, the photocurrent decreased gradually. With high BiOI concentration, more BiOI was immobilized on the electrode surface, which can hinder the electron transfer and increase the recombination of electrons and holes, resulting in a decreased photocurrent. Thus, 4 mg/mL BiOI was adopted to construct the biosensor. As a typical cross-linking agent, the modified amount of MIPA can also influence the fabrication of biosensor and the performance of the biosensor. Therefore, optimization of the incubation time of MIPA is extremely critical. As seen in Fig. 4B, with extending the incubation from 20 to 100 min, the photocurrent decreased successively, which could be ascribed to the increase in the immobilized amount of MIPA on the electrode surface. However, with further lengthening the incubation time, the photocurrent tended to level off, which might be caused by the saturated immobilization of MIPA. Considering the detection efficiency, 100 min was employed as MIPA incubation time. As the detection target, the capture of CoA is crucial. Thus, the reaction time for CoA and MIPA were optimized. The result is illustrated in Fig. 4C. the photocurrent decreased with prolonging the reaction time to 100 min, and then the photocurrent almost not change. Thus, 100 min was chosen as the optimal reaction time. In addition, the immobilization time of ZnO quantum dots were also optimized. According to the results in Fig. 4D, 100 min was used.
Fig. 3. PEC response of different electrodes in 0.1 M PBS containing 0.1 M AA (pH = 7.4). (a) BiOI/ITO, (b) Fe3O4/BiOI/ITO, (c) MIPA/Fe3O4/BiOI/ITO, (d) CoA/MIPA/Fe3O4/BiOI/ITO, (e) ZnO/CoA/MIPA/Fe3O4/BiOI/ITO.
when MIPA was immobilized onto the electrode surface, the photocurrent decreased (curve c), which could be explained as the steric hindrance effect, hindering the diffusion of the electron donor to electrode surface. Afterwards, based on the covalent attachment of the thiol group of CoA to the maleimide group of MIPA, CoA was trapped on the electrode surface, leading to a suppression of the transfer rate of photogenerated electrons due to the negatively charged phosphate of CoA. Thus, the photoelectrochemical response decreased (curve d) [43]. Subsequently, when ZnO-QDs were modified on the electrode surface, the photocurrent further decreased (curve e). It can be explained that the ZnO-QDs have a stronger light absorption capability and compete to consume electron donor [44].
3.5. Histone acetyltransferase activity assay Under optimal conditions, the performance of the biosensor was investigated using a wide range of HAT concentration. As described in
Fig. 4. Effect of different experimental factors on the response of the photoelectrochemical biosensor. (A) BiOI concentration, (B) MIPA incubation time, (C) CoA reaction time and (D) ZnO quantum dots immobilization time in 0.1 M PBS containing 0.1 M AA (pH = 7.4). 5
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Fig. 5. (A) Photoelectrochemical response of the biosensor with different concentrations of HAT. a–k, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 200, 500 nM. (B) Calibration curve of the biosensor for HAT detection. (C) The histogram for the photocurrent changes of the biosensor fabricated with different targets. The concentration of different targets was 100 nM. (D) Time-based photocurrent response of the biosensor toward 100 nM HAT.
low detection limit, indicating that the photoelectrochemical biosensor has great potential and prospects for detecting HAT. Since selectivity was an indispensable factor for biosensors, the selectivity of the biosensor was investigated using HRP, ALP, PKA, BSA as possible interferers. To assess the detection selectivity, the photocurrent changes for different interferers (ΔI = I2 - I1) were compared, where I2 was the photocurrent of MIPA/Fe3O4/BiOI/ITO, and I1 was the photocurrent of biosensor with different interferers. As can be seen from Fig. 5C, the difference for ΔI is obvious, which indicates that the good selectivity of the developed method. For the practicality of the sensor, in addition to its sensitivity and specificity to be guaranteed, stability is also a part that cannot be ignored. Therefore, stability was investigated and the results were shown in Fig. 5D. It is worth noting that the photocurrent response values of the seven cycles are almost identical, and the calculated relative standard deviation (RSD) is 0.92 %, showing superior stability.
Table 1 Performance comparison of the photoelectrochemical biosensor with other methods for HAT detection. Methods
Linear range
Detection limit
References
Electrochemiluminescence Electrochemiluminescence Time-resolved luminescence Fluorescence Fluorescence Fluorescence Colorimetry Electrochemistry Electrochemistry Photoelectrochemistry
0.1−100 nM 0.1−100 nM 0.2−100 nM 0.1-120 nM 0.5−100 nM 0.5−100 nM 1-200 nM 1−500 nM 0.1−100 nM 0.01−500 nM
0.074 nM 0.05 nM 0.05 nM 0.05 nM 0.2 nM 0.1 nM 0.2 nM 0.1 nM 0.067 nM 3.3 pM
[6] [5] [12] [45] [13] [10] [13] [46] [11] This work
Fig. 5A, the photocurrent enhanced gradually with increasing HAT concentration from 0.01 to 500 nM. An excellent linear relationship between the photocurrent intensity and the logarithm value of HAT concentration can be obtained with the linear regression equation of I (μA) = - 0.27logc (nM) + 2.17 (R2 = 0.9988). The detection limit was 0.003 nM (S/N = 3) (Fig. 5B). Compared with other methods (Table 1), this photoelectrochemical method possesses wide detection range and
3.6. Inhibition assay HAT plays a vital role in epigenetics and plays a non-negligible role. The abnormal activity of HAT is closely related to the occurrence of disease [3]. Thus, the research of effective inhibitors of HAT is urgent
Fig. 6. Diagram of the relationship between change ratio and antibiotic (tobramycin (A), amoxicillin (B)) concentration. a–i, 1, 2, 5, 10, 30, 40, 50 μM (the concentration of p300 was 100 nM). 6
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for the treatment of HAT-related diseases. To further demonstrate the general applicability of this method, the effects of antibiotics on the catalytic activity of HAT were investigated. Here, tobramycin and amoxicillin were conducted as antibiotic models. As can be seen from Fig. 6, antibiotics successfully inhibited the activity of histone acetyltransferase. The IC50 (half maximal inhibitory concentration) of tobramycin and amoxicillin were calculated to be 3.87 and 10.44 μM, respectively. At present, the abuse of antibiotics in China is becoming increasingly serious, and the excessive use of antibiotics will cause resistance to humans, animals and plants, so it is extremely necessary to study the ecological toxicological effect of antibiotics and the impact of antibiotics on enzyme activity. Therefore, it is helpful to evaluate the hazards of the overuse of antibiotics and develop precautions for the use of antibiotics.
[11]
[12]
[13]
[14]
[15] [16]
4. Conclusion [17]
In conclusion, an advanced quantum dot signal amplification PEC sensor was constructed for histone acetyltransferase detection, based on the specific recognition of thiol groups in coenzyme A by maleimide and the competitive electron donor of ZnO-QDs. The proposed sensor detects histone acetyltransferase as low as ∼3.3 pM with a linear range of 0.01–500 nM, and can convenient readout of assay via photocurrent response according to the standard curve. More importantly, the strategy is not only applied to the detection of histone acetyltransferase, but also can be extended to medical diagnosis due to the ecotoxicological study of antibiotics. This strategy will supply a foothold for the future prosperity of various PEC biological analysis methods based on histone acetyltransferases, and representing the prospect and potential that cannot be ignored in the field of medical diagnosis.
[18]
[19]
[20]
[21]
[22]
Declaration of Competing Interest
[23]
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
[24]
[25]
Acknowledgments
[26]
This work was supported by the National Natural Science Foundation of China (Nos. 21775090, 41807484), the Natural Science Foundation of Shandong Province of China (Nos. ZR2018MB028).
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Yunlei Zhou received doctor degree in College of Life Science, Beijing Normal University in 2013. Now she is an associate Professor in College of Chemistry and Material Science, Shandong Agricultural University. Her current interest is electrochemical biosensor. Huanshun Yin received doctor degree in Soil Science in College of Resources and Environment, Shandong Agricultural University in 2012. Now he is a Professor in College of Chemistry and Material Science, Shandong Agricultural University. His current interest is electrochemical bioassay. Fei Li received her bachelor’s degree in material chemistry from College of Chemistry and Material Science, Shandong Agricultural University in 2018. Now, she is a postgraduate student of College of Chemistry and Material Science, Shandong Agriculture University. Her current interest is electrochemical biosensor. Hui Li is a college student of College of Chemistry and Material Science, Shandong Agricultural University. Runze Guo is a college student of College of Chemistry and Material Science, Shandong Agricultural University. Yihan Han is a college student of College of Chemistry and Material Science, Shandong Agricultural University. Shiyun Ai is a professor in College of Chemistry and Material Science, Shandong Agricultural University. He received his doctor degree (analytical chemistry) from Department of Chemistry, East China Normal University in 2004. His current research interests include bioelectroanalysis and preparation of nano-functional material.
Yan Chen received her bachelor’s degree in material chemistry from College of Chemistry and Material Science, Shandong Agricultural University in 2017. Now, she is a postgraduate student of College of Chemistry and Material Science, Shandong Agriculture University. Her current interest is electrochemical biosensor.
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Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Photoelectrochemical biosensor for histone acetyltransferase detection based on ZnO quantum dots inhibited photoactivity of BiOI nanoflower
T
Yan Chen, Yunlei Zhou*, Huanshun Yin, Fei Li, Hui Li, Runze Guo, Yihao Han, Shiyun Ai College of Chemistry and Material Science, Shandong Agricultural University, 271018, Taian, Shandong, People’s Republic of China
A R T I C LE I N FO
A B S T R A C T
Keywords: Histone acetylation Photoelectrochemical biosensor BiOI nanoflower Antibiotic ZnO quantum dots
Histone acetylation is a very important post-transcriptional modification, which is catalyzed by histone acetyltransferase with acetyl group donor of acetyl coenzyme A (Ac-CoA). Abnormal histone acetylation is associated with many diseases, so it is important to develop a sensitive and efficient method for detecting histone acetyltransferase (HAT). In this paper, a simple and sensitive photoelectrochemical biosensor was established for indirect detection of HAT using the acetylation reaction by-product of coenzyme A (CoA) as target molecule. To construct the biosensor, BiOI nanoflower was employed as photoactive material, Fe3O4-NH2 and 3-maleimidopropionic acid were used as “bridge” reagents for immobilizing CoA, and ZnO quantum dots were adopted as photocurrent inhibitor. Under optimal experimental conditions, the photocurrent decreased with changing HAT concentration from 0.01 to 500 nM. The linear relationship between the photocurrent and the logarithm value of HAT concentration can be constructed with this concentration range and the detection limit was 3 pM (S/N = 3). The sensor has excellent detection specificity, good stability and reproducibility. Based on the current situation of serious antibiotic contamination, the effect of antibiotics on HAT activity was investigated using this photoelectrochemical biosensor.
1. Introduction Histone acetylation is an important covalent post-translational epigenetic modification, which plays crucial role in transcription, DNA replication, DNA damage/repair, and histone deposition [1]. Histone acetylation is catalyzed by histone acetyltransferase (HAT), which transfers acetyl group from acetyl donor of acetyl coenzyme A (Ac-CoA) to lysine residue of histone [2]. In this catalysis process, the donor of Ac-CoA is then changed to coenzyme A (CoA). It has been reported that histone acetylation has a significant effect on neurological disorders and is associated with metabolic syndrome, chronic inflammation, HIV infection, as well as cancer [3]. To further understand the biological functions of histone acetylation, the possible indication effect on diseases and relevant drug screening, the determination of histone acetylation and HAT with simple, sensitive and selective method is necessary. Traditional technique for HAT detection mainly relied on radioisotope labeling [4], which is limited due to the high risk to human health under radioactive exposure. Thus, various alternative techniques have been developed, including electrochemiluminescence [5–7], fluorescence [8–10], electrochemistry [11], time-resolved luminescence and [12] colorimetry [13]. Some of them depend on antibody and ⁎
enzyme; some of them depend on the resistance of peptide to protease hydrolysis and the charge properties with and without acetylation; some of them depend on the by-product (CoA) of acetylation process. Though these methods achieve the sensitive and selective detection of HAT, new method is also highly desirable with merits of simple operation, rapid response, inexpensive instrument, high sensitivity and specificity. Photoelectrochemical technique, which combines the advantages of optical method and electrochemical method, attracts wide attention because of its low cost, simple operation, low background signal and high sensitivity [14]. Up to now, various fields including DNA [15], microRNA [16], protein [17], small biomolecules [18], metal ions [19] have been infiltrated by photoelectrochemical sensors, which strongly demonstrates the widely applicability of photoelectrochemical detection platform. However, the application of photoelectrochemical biosensor for HAT detection is missed. As one of the important parameters for PEC biosensor, the origin of the photoelectrochemical signal may greatly influence the detection sensitivity, which mainly depends on the photoactive material. In recent years, various semiconductor materials including ZnO [20,21], TiO2 [22], C3N4 [23], BiOI [24] have been applied to the construction of PEC sensors. Among them, BiOI has attracted widespread concern due to its narrow band gap (1.8 eV) [25]
Corresponding author. E-mail address: [email protected] (Y. Zhou).
https://doi.org/10.1016/j.snb.2019.127633 Received 5 November 2019; Received in revised form 23 December 2019; Accepted 24 December 2019 Available online 28 December 2019 0925-4005/ © 2019 Elsevier B.V. All rights reserved.
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irradiation source. To eliminate the ultraviolet light, an optical filter was equipped on the Xe lamp source. Three-electrode system was employed, where ITO or modified ITO electrode was used as working electrode, saturated calomel electrode was used as reference electrode, and platinum electrode was used as counter electrode. The applied potential is -0.4 V.
and strong visible light absorption ability, showing excellent optical and electrical properties [26]. For examples, Zhou et al. developed a sensitive photoelectrochemical biosensor for DNA methyltransferase activity assay using BiOI as a photoactive material [27]; Gong et al. successfully implemented biofunctional crossed BiOI flake arrays to achieve highly sensitive photoelectric biosensor for organophosphate pesticide [18]. In view of the excellent feature of photoactivity, BiOI is promising in the fabrication of photoelectrochemical biosensor with superior performances. Recently, ″signal-off″ model photoelectrochemical biosensor has attracted more attentions. As a kind of photoactive material, quantum dots (QDs) received widely applications in photoelectrochemical biosensor [28,29]. In view of the QDs has many unique physical effects, such as good biocompatibility, strong stability, leading to its great applications in photoelectric materials, biofluorescence labeling and so on [30]. Among various QDs, ZnO QDs attract our attentions due to the reaction activity of Zn2+ with phosphate group and the inhibition activity of ZnO to other photoactive materials [31,32]. As a kind of byproduct of histone acetylation, CoA has phosphate group, which can facilitate the capture of ZnO QDs to quench the photoelectrochemical signal. In this work, a simple, sensitive and selective photoelectrochemical method was developed for indirect HAT detection. As the by-product of the acetylation reaction catalyzed by HAT, CoA was employed as detection target molecule. BiOI nanoflowers were used as photoactive material and ZnO-QDs were used as photoelectrochemical signal inhibitor. To evaluate the applicability of the developed method, the effect of antibiotics on HAT activity was investigated, which may provide new mechanism on ecotoxicological effect antibiotics, offer new biomarker and new technique on ecotoxicological effect evaluation.
2.2. Synthesis of BiOI nanoflower BiOI nanoflower was prepared according to previous report [33]. In general, 1.94 g of Bi(NO3)3 was dissolved in 70 mL of ethylene glycol, followed by the addition of 0.664 g of KI. The ratio of n(Bi(NO3)3 and n (KI) was 1:1. After completely dissolved under magnetic stirring, the solution was transferred into a Teflon-lined autoclave and heated at 160 °C for 12 h. After the autoclave was cooled down naturally to room temperature, the precipitate was separated by centrifuging at 4000 rom for 10 min, which was then washed with water and ethanol for three times. Finally, the prepared BiOI was dried at 60 °C. 2.3. Preparation of Fe3O4-NH2 Fe3O4 was prepared based on previous paper by slightly modifying the previous paper [34]. In summary, 4.0 g FeCl2·4H2O and 9.5 g FeCl3·6H2O were dissolved in a three-necked flask containing 110 mL of deionized water and filled with N2 to eliminate the effect of O2. Then, 20 mL of 25 % ammonia water was added into the reaction system, which was then heated to 90 °C for 30 min. The precipitate was collected by the separation of magnet with washing with water and ethanol for several times. Finally, the prepared Fe3O4 was dried at 60 °C. To prepare Fe3O4-NH2, the obtained Fe3O4 and 200 mL ethanol were added into a three-necked flask under N2 atmosphere. The dispersion was heated to 75 °C. Then, 5 mL of 95 % APTES was further poured into the reaction system. After magnetic stirring for 2 h, the precipitate was separated by a magnet, which was then washed with water and ethanol for several times. Finally, the prepared Fe3O4-NH2 was dried at 60 °C.
2. Experimental section 2.1. Reagents and apparatus Horseradish peroxidase (HRP), alkaline phosphatase (ALP), bovine serum albumin (BSA) were obtained from Sigma-Aldrich (USA). Protein kinase A (PKA) was provided by NEB (USA). Histone acetyltransferase p300 (HAT) was supplied by Enzo. Biochem. (USA). The substrate peptide with the specific sequence of RGKGGKGLGKGGAKA was synthesized by Sangon (China). Bi(NO3)3, KI, FeCl2·4H2O, FeCl3·6H2O, NaH2PO4, Na2HPO4, NH3·H2O, Zn(CH3COO)2▪2H2O, CH3OH, disodium ethylenediaminetetraacetic acid (EDTA), (3-Aminopropyl)triethoxysilane (APTES), Ac-CoA (Enzymatic ≥ 83 %), CoA, tris (2-carboxyethyl)phosphine (TCEP), tris(hydroxymethyl) aminomethane (Tris), tobramycin, amoxicillin and tetracycline were purchased from Aladdin (China). A 96-well detachable ELISA plate was supplied by Corning Costa (USA). ITO conductive glass was purchased from Kaivo Electronic Components Co., Ltd. (Zhuhai, China, ITO coating 180 ± 25 nautical miles, sheet resistance < 15 Ω/cm2). Other unspecified reagents were of analytical grade and were not further purified. The buffer solutions used in the experiment were as follows. Washing buffer: 10 mM Tris−HCl, 50 mM NaCl (pH 7.4). HAT reaction buffer: 10 mM Tris−HCl, 50 mM NaCl and 10 mM MgCl2 (pH 7.4). Photoelectrochemical detection buffer: 0.1 M PBS containing 0.1 M AA (pH = 7.4). All solutions were prepared using distilled deionized water produced by DEPC. Scanning electron microscopy (SEM) images were recorded by Hitachi S-4800 SEM (Japan). JEM-2100 microscope (Japan) was used to measure transmission electron microscopy (TEM) images. Fourier transform infrared spectroscopy (FT-IR) was determined by Thermo Nicolet-380 IR spectrophotometer (USA) in the range of 4000 – 400 cm−1. The crystal structure of the material was analyzed by X-ray diffraction (XRD, Cu Kα, Rigaku, D/max 2500, Japan) at room temperature. Photoelectrochemical measurement was performed on a CHI832A electrochemical station with a 500 W Xe lamp as the
2.4. Synthesis of ZnO QDs ZnO QDs were prepared according to previous work using ZnO nanospheres as precursor [32,35]. Briefly, 0.1 M zinc acetate dihydrate was dissolved into 50 mL of methanol under magnetic stirring. After that, 25 mL of 0.5 M NaOH solution in methanol was further added into the above solution. After magnetic stirring for 30 min, the solution was then poured into a Teflon-lined stainless-steel autoclave and heated at 150 °C for 6 h. After naturally cooled to room temperature, the sediment was collected by centrifugation, which was washed with water and ethanol for several times. The prepared ZnO nanospheres was dried at 60 °C. 7.5 mg ZnO nanospheres was dispersed into 15 mL deionized water with the aid of ultrasonication for 1 h. Then, 1 mL of 0.1 M ascorbic acid aqueous solution was further added into the dispersion, which was transferred to a Teflon-line stainless steel autoclave and heated at 200 °C for 2 h. After cooling to room temperature naturally, it was centrifuged at 4000 rpm for 10 min to remove impurities, and the dispersion was collected. Finally, the dispersion was poured into a dialysis membrane for 12 h of dialysis. 2.5. Acetylation reaction Firstly, 30 μL 2 × HAT reaction buffer containing different concentration of HAT (p300) and 20 μM Ac-CoA were added into a centrifugal tube. Then, 10 μL of 6 μM substrate peptide was further added into the centrifugal tube. The centrifugal tube was oscillated for two hours in humid environment at 37 °C. Afterwards, the solution was 2
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catalysis process, acetyl group of Ac-CoA can be transferred to the lysine residue of substance peptide, and CoA can be produced as a byproduct. CoA can be easily recognized and captured due to the thiol group and phosphate group in its structure. Thus, CoA was employed as target molecule to achieve the indirect detection of HAT. Fig. 1B illustrates the fabrication of photoelectrochemical biosensor and HAT detection. Firstly, BiOI was immobilized on the bare ITO electrode surface as photoactive material. Then, Fe3O4-NH2 was modified to provide –NH2 on the electrode surface. Afterwards, based on the covalent reaction between –NH2 and −COOH [36], 3-maleimidopropionic acid (MIPA) was further modified on the electrode surface, which could easily reacted with thiol group [37,38] Thus, CoA was then captured and its phosphate group was away from the electrode surface. Finally, based on the specific interaction between ZnO and phosphate group [39], ZnO QDs were modified on the electrode surface. According to the change of the photocurrent, HAT can be detected.
transferred to a 96-well removable ELISA plate and incubated for 30 min, after which the supernatant was transferred to a centrifugal tube and stored at 4 °C before use (the resulting solution was designated as Target Solution). 2.6. Fabrication of the biosensor The ITO conductive glass (1 × 5 cm) was pretreated with the aid of ultrasonication with acetone and ethanol/NaOH solution (v/v, 1:1), respectively. Then, the electrode was rinsed with double distilled water and dried naturally at room temperature. After that, 40 μL of 4 mg/mL BiOI dispersion was dropped onto the electrode surface, and dried under the irradiation of an infrared lamp (The electrode was named BiOI/ITO). Subsequently, 40 μL of 0.5 mg/mL Fe3O4-NH2 dispersion was further casted onto the electrode surface and dried under the irradiation of infrared lamp (The obtained electrode was named Fe3O4/ BiOI/ITO). After that, 40 μL of 2 mg/mL 3-maleimidopropionic acid (MIPA) was dropped onto Fe3O4/BiOI/ITO electrode surface and incubated for 120 min in a humidified environment at 37 °C, where the 3maleimide propionic acid was activated by 1 μM EDC and 1 μM NHS before use. The prepared electrode was denoted as MIPA/Fe3O4/BiOI/ ITO. Afterwards, the electrode was incubated with 40 μL Target Solution (described in section 2.5) for 120 min in a humid cell at 37 °C. The obtained electrode was labeled as CoA/MIPA/Fe3O4/BiOI/ITO. Finally, the electrode was incubated with 40 μL ZnO QDs under humid conditions for 120 min. The prepared electrode was named as ZnO/ CoA/MIPA/Fe3O4/BiOI/ITO. After each modification process, the electrode was rinsed three times with washing buffer.
3.2. Characterization of nanomaterials The morphology and elemental distribution of BiOI were analyzed by SEM and mapping, respectively. As depicted in Fig. 1A, the prepared BiOI was a flower-like spherical structure with an average diameter of about 2 μm, which was stacked by a plurality of staggered sheet structures with thickness of 10−20 nm. The distribution of elements in the BiOI was further determined by EDS. The results indicate that the elements of the prepared material are evenly distributed. The morphology of Fe3O4-NH2 was investigated by TEM, which showed a relatively uniform distribution, but slightly agglomerated, and the particle size was between 12 and 16 nm (Fig. 1C). Fig. 1D presented the TEM and HRTEM images of ZnO QDs. The lattice spacing of 0.26 nm corresponds exactly to the (002) crystal plane of ZnO (JCPDS No. 36–1451) [11], indicating the successful preparation of ZnO QDs. XRD was employed to further explore the crystal configuration of synthetic BiOI. As illustrated in Fig. 2A, the crystal structure of BiOI corresponds exactly to JCPDS card No. 10-0445, indicating the successful preparation of BiOI with a standard tetragonal phase [40]. The XRD patterns of Fe3O4 and Fe3O4-NH2 were depicted in Fig. 2B. The positions of the diffraction peaks are basically consistent before and after amino modification, indicating that the amino modification does not change the crystal form of Fe3O4, but the diffraction peak was slightly reduced, which could be attributed to the reduction of the crystallinity of Fe3O4 by amino modification [3]. In order to more accurately understand the successful modification of the amino group, the FT-IR was measured (Fig. 2C). The absorption peak at 588 cm−1 was derived from the stretching vibration of Fe-O, demonstrating the successful synthesis of the Fe3O4 nanospheres (black line). After treatment with APTES, the absorption peaks of 3135 and 1105 cm−1 was assigned to the stretching vibration of NeH bond and stretch vibration of Si-O bond, respectively. These results prove the successful amino-
2.7. Effect of antibiotic on HAT activity assay The effects of antibiotics on HAT activity were assessed. To perform it, different concentrations of antibiotics (tobramycin and amoxicillin) were added to the acetylation reaction system as described in Section 2.5, followed by the construction of the biosensor as described in Section 2.6. The results were evaluated by comparing the photocurrent change ratio (PCR (%) = (I1 – I2)/I2) of the biosensor with and without antibiotic, where I1 represented the photocurrent of ZnO/CoA/MIPA/ Fe3O4/BiOI/ITO with agents, and I2 was the photocurrent of ZnO/CoA/ MIPA/Fe3O4/BiOI/ITO without agents. 3. Results and discussion 3.1. Detection strategy In this paper, a photoelectrochemical biosensor was developed for HAT detection based on the by-product of CoA in the acetylation process of peptide catalyzed by HAT. In this detection strategy, the indirect HAT detection can be achieved using CoA as detection target. As shown in Scheme 1A, the amount of CoA is related to HAT activity. In the
Scheme 1. (A) Mechanism of the acetylation of short peptide catalyzed by HAT. (B) Schematic diagram of the construction of PEC biosensor for detecting HAT. 3
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Fig. 1. SEM image (A) and mapping image (B) of BiOI. Inset: SEM image of partially enlarged BiOI. TEM image of Fe3O4-NH2 (C). TEM and HRTEM (inset) image of ZnO Quantum Dots (D).
electrodes were experimentally determined and contrasted. As clearly shown in Fig. 3, a strong photocurrent was produced when BiOI was attached onto the bare ITO electrode surface (curve a), indicating that the excellent photoactivity of BiOI [24,41]. After the modification of Fe3O4-NH2 to the BiOI/ITO surface (curve b), the photocurrent intensity significantly increased, which may be attributed not only to the good electrical conductivity of Fe3O4, but also due to the heterojunction formed by the synergistic effect of Fe3O4 and BiOI, which increased the absorption of visible light, facilitating the generation, transfer and the separation of photo-generated electrons and holes [40,42]. Afterwards,
functionalization of Fe3O4 (red line) [3]. FT-IR was also employed to characterize ZnO QDs. As displayed in Fig. 2D, the peak at 470 cm−1 belonged to the Zn-O bond, which was the characteristic peak of ZnO [4]. Integrating the results in Figs. 1D and 2 D, ZnO QDS had been successful prepared.
3.3. Detection feasibility assay In order to verify the practicability of the prepared photoelectrochemical biosensor, the photocurrent responses of different
Fig. 2. XRD patterns of BiOI (A), Fe3O4 and Fe3O4-NH2 (B). (C) FT-IR image of Fe3O4 (black line) and Fe3O4-NH2 (red line). (D) FT-IR image of ZnO QDs (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.). 4
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3.4. Optimization of experimental conditions In order to maximize the performance of the biosensor, several influencing factors were optimized, including BiOI concentration, MIPA incubation time, CoA reaction time and ZnO quantum dots immobilization time. As shown in Fig. 4A, the photocurrent intensity enhanced with changing BiOI concentration from 0.5 to 4 mg/mL. With increasing BiOI concentration, more photogenerated electrons produced, which led to the increased photocurrent. However, when further increasing BiOI concentration, the photocurrent decreased gradually. With high BiOI concentration, more BiOI was immobilized on the electrode surface, which can hinder the electron transfer and increase the recombination of electrons and holes, resulting in a decreased photocurrent. Thus, 4 mg/mL BiOI was adopted to construct the biosensor. As a typical cross-linking agent, the modified amount of MIPA can also influence the fabrication of biosensor and the performance of the biosensor. Therefore, optimization of the incubation time of MIPA is extremely critical. As seen in Fig. 4B, with extending the incubation from 20 to 100 min, the photocurrent decreased successively, which could be ascribed to the increase in the immobilized amount of MIPA on the electrode surface. However, with further lengthening the incubation time, the photocurrent tended to level off, which might be caused by the saturated immobilization of MIPA. Considering the detection efficiency, 100 min was employed as MIPA incubation time. As the detection target, the capture of CoA is crucial. Thus, the reaction time for CoA and MIPA were optimized. The result is illustrated in Fig. 4C. the photocurrent decreased with prolonging the reaction time to 100 min, and then the photocurrent almost not change. Thus, 100 min was chosen as the optimal reaction time. In addition, the immobilization time of ZnO quantum dots were also optimized. According to the results in Fig. 4D, 100 min was used.
Fig. 3. PEC response of different electrodes in 0.1 M PBS containing 0.1 M AA (pH = 7.4). (a) BiOI/ITO, (b) Fe3O4/BiOI/ITO, (c) MIPA/Fe3O4/BiOI/ITO, (d) CoA/MIPA/Fe3O4/BiOI/ITO, (e) ZnO/CoA/MIPA/Fe3O4/BiOI/ITO.
when MIPA was immobilized onto the electrode surface, the photocurrent decreased (curve c), which could be explained as the steric hindrance effect, hindering the diffusion of the electron donor to electrode surface. Afterwards, based on the covalent attachment of the thiol group of CoA to the maleimide group of MIPA, CoA was trapped on the electrode surface, leading to a suppression of the transfer rate of photogenerated electrons due to the negatively charged phosphate of CoA. Thus, the photoelectrochemical response decreased (curve d) [43]. Subsequently, when ZnO-QDs were modified on the electrode surface, the photocurrent further decreased (curve e). It can be explained that the ZnO-QDs have a stronger light absorption capability and compete to consume electron donor [44].
3.5. Histone acetyltransferase activity assay Under optimal conditions, the performance of the biosensor was investigated using a wide range of HAT concentration. As described in
Fig. 4. Effect of different experimental factors on the response of the photoelectrochemical biosensor. (A) BiOI concentration, (B) MIPA incubation time, (C) CoA reaction time and (D) ZnO quantum dots immobilization time in 0.1 M PBS containing 0.1 M AA (pH = 7.4). 5
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Fig. 5. (A) Photoelectrochemical response of the biosensor with different concentrations of HAT. a–k, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, 200, 500 nM. (B) Calibration curve of the biosensor for HAT detection. (C) The histogram for the photocurrent changes of the biosensor fabricated with different targets. The concentration of different targets was 100 nM. (D) Time-based photocurrent response of the biosensor toward 100 nM HAT.
low detection limit, indicating that the photoelectrochemical biosensor has great potential and prospects for detecting HAT. Since selectivity was an indispensable factor for biosensors, the selectivity of the biosensor was investigated using HRP, ALP, PKA, BSA as possible interferers. To assess the detection selectivity, the photocurrent changes for different interferers (ΔI = I2 - I1) were compared, where I2 was the photocurrent of MIPA/Fe3O4/BiOI/ITO, and I1 was the photocurrent of biosensor with different interferers. As can be seen from Fig. 5C, the difference for ΔI is obvious, which indicates that the good selectivity of the developed method. For the practicality of the sensor, in addition to its sensitivity and specificity to be guaranteed, stability is also a part that cannot be ignored. Therefore, stability was investigated and the results were shown in Fig. 5D. It is worth noting that the photocurrent response values of the seven cycles are almost identical, and the calculated relative standard deviation (RSD) is 0.92 %, showing superior stability.
Table 1 Performance comparison of the photoelectrochemical biosensor with other methods for HAT detection. Methods
Linear range
Detection limit
References
Electrochemiluminescence Electrochemiluminescence Time-resolved luminescence Fluorescence Fluorescence Fluorescence Colorimetry Electrochemistry Electrochemistry Photoelectrochemistry
0.1−100 nM 0.1−100 nM 0.2−100 nM 0.1-120 nM 0.5−100 nM 0.5−100 nM 1-200 nM 1−500 nM 0.1−100 nM 0.01−500 nM
0.074 nM 0.05 nM 0.05 nM 0.05 nM 0.2 nM 0.1 nM 0.2 nM 0.1 nM 0.067 nM 3.3 pM
[6] [5] [12] [45] [13] [10] [13] [46] [11] This work
Fig. 5A, the photocurrent enhanced gradually with increasing HAT concentration from 0.01 to 500 nM. An excellent linear relationship between the photocurrent intensity and the logarithm value of HAT concentration can be obtained with the linear regression equation of I (μA) = - 0.27logc (nM) + 2.17 (R2 = 0.9988). The detection limit was 0.003 nM (S/N = 3) (Fig. 5B). Compared with other methods (Table 1), this photoelectrochemical method possesses wide detection range and
3.6. Inhibition assay HAT plays a vital role in epigenetics and plays a non-negligible role. The abnormal activity of HAT is closely related to the occurrence of disease [3]. Thus, the research of effective inhibitors of HAT is urgent
Fig. 6. Diagram of the relationship between change ratio and antibiotic (tobramycin (A), amoxicillin (B)) concentration. a–i, 1, 2, 5, 10, 30, 40, 50 μM (the concentration of p300 was 100 nM). 6
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for the treatment of HAT-related diseases. To further demonstrate the general applicability of this method, the effects of antibiotics on the catalytic activity of HAT were investigated. Here, tobramycin and amoxicillin were conducted as antibiotic models. As can be seen from Fig. 6, antibiotics successfully inhibited the activity of histone acetyltransferase. The IC50 (half maximal inhibitory concentration) of tobramycin and amoxicillin were calculated to be 3.87 and 10.44 μM, respectively. At present, the abuse of antibiotics in China is becoming increasingly serious, and the excessive use of antibiotics will cause resistance to humans, animals and plants, so it is extremely necessary to study the ecological toxicological effect of antibiotics and the impact of antibiotics on enzyme activity. Therefore, it is helpful to evaluate the hazards of the overuse of antibiotics and develop precautions for the use of antibiotics.
[11]
[12]
[13]
[14]
[15] [16]
4. Conclusion [17]
In conclusion, an advanced quantum dot signal amplification PEC sensor was constructed for histone acetyltransferase detection, based on the specific recognition of thiol groups in coenzyme A by maleimide and the competitive electron donor of ZnO-QDs. The proposed sensor detects histone acetyltransferase as low as ∼3.3 pM with a linear range of 0.01–500 nM, and can convenient readout of assay via photocurrent response according to the standard curve. More importantly, the strategy is not only applied to the detection of histone acetyltransferase, but also can be extended to medical diagnosis due to the ecotoxicological study of antibiotics. This strategy will supply a foothold for the future prosperity of various PEC biological analysis methods based on histone acetyltransferases, and representing the prospect and potential that cannot be ignored in the field of medical diagnosis.
[18]
[19]
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Declaration of Competing Interest
[23]
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Acknowledgments
[26]
This work was supported by the National Natural Science Foundation of China (Nos. 21775090, 41807484), the Natural Science Foundation of Shandong Province of China (Nos. ZR2018MB028).
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Yunlei Zhou received doctor degree in College of Life Science, Beijing Normal University in 2013. Now she is an associate Professor in College of Chemistry and Material Science, Shandong Agricultural University. Her current interest is electrochemical biosensor. Huanshun Yin received doctor degree in Soil Science in College of Resources and Environment, Shandong Agricultural University in 2012. Now he is a Professor in College of Chemistry and Material Science, Shandong Agricultural University. His current interest is electrochemical bioassay. Fei Li received her bachelor’s degree in material chemistry from College of Chemistry and Material Science, Shandong Agricultural University in 2018. Now, she is a postgraduate student of College of Chemistry and Material Science, Shandong Agriculture University. Her current interest is electrochemical biosensor. Hui Li is a college student of College of Chemistry and Material Science, Shandong Agricultural University. Runze Guo is a college student of College of Chemistry and Material Science, Shandong Agricultural University. Yihan Han is a college student of College of Chemistry and Material Science, Shandong Agricultural University. Shiyun Ai is a professor in College of Chemistry and Material Science, Shandong Agricultural University. He received his doctor degree (analytical chemistry) from Department of Chemistry, East China Normal University in 2004. His current research interests include bioelectroanalysis and preparation of nano-functional material.
Yan Chen received her bachelor’s degree in material chemistry from College of Chemistry and Material Science, Shandong Agricultural University in 2017. Now, she is a postgraduate student of College of Chemistry and Material Science, Shandong Agriculture University. Her current interest is electrochemical biosensor.
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