One-step fabrication of trimetallic alloy nanozyme catalyzer for luminol-H2O2 chemiluminescence and its application for miRNA-21 detection coupled with miRNA walking machine

Journal of Pharmaceutical and Biomedical Analysis 186 (2020) 113280

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One-step fabrication of trimetallic alloy nanozyme catalyzer for luminol-H2 O2 chemiluminescence and its application for miRNA-21 detection coupled with miRNA walking machine Shuyu Mei a , Bingru Liu b , Xiaoli Xiong b , Xu Hun b,∗ a

Department of Pathology, Tianjin Bao Di Hospital, Bao Di Clinical College of Tianjin Medical University, Tianjin, 301800, China Key Laboratory of Optic-electric Sensing and Analytical Chemistry for Life Science, MOE, Shandong Key Laboratory of Biochemical Analysis, Key Laboratory of Analytical Chemistry for Life Science in Universities of Shandong, State Key Laboratory Base of Eco-chemical Engineering, Key Laboratory of Rubber-Plastics of Ministry of Education/Shandong Provincial Key Laboratory of Rubber Plastics, College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao, 266042, China b

a r t i c l e

i n f o

Article history: Received 20 January 2020 Received in revised form 21 March 2020 Accepted 22 March 2020 Available online 26 March 2020 Keywords: Trimetallic alloys miRNA-21 Enzyme-free One-step Nanocatalyst

a b s t r a c t PtCuCo trimetallic alloys (PtCuCo-TAs) are synthesized by one-step reduction. The chemiluminescence (CL) properties of PtCuCo-TAs are studied systemically. PtCuCo-TAs show good catalyzing for luminolH2 O2 system. A CL platform is developed for the detection of miRNA-21 using PtCuCo-TAs as nanozyme catalyzer. In the CL detection platform, H1 (Hairpin DNA1) is immobilized onto magnetic beads (MBs) firstly. In the presence of miRNA-21, H1 is opened. H2 (Hairpin DNA2) then hybridizes with H1. Meanwhile, a “cleat” in the end of miRNA-21 with a fewer bases complementary is formed to prevent miRNA-21 dissociating from H1. This miRNA-21 hybridizes to another H1. When cpDNA-PtCuCo-TAs which consisted with cDNA (Complementary strand of probe DNA) and pDNA-PtCuCo-TAs (PtCuCo-TAs labeled with probe DNA) are added, the ssDNA region of H1 reacts with the toehold domain of probe DNA and cDNA is released resulting pDNA-PtCuCo-TAs being captured. With this process repeatedly, a lot of pDNAPtCuCo-TAs are captured onto MBs. After separation and washing, the precipitate and H2 O2 are put into the 96-well and luminol solution is injected. The CL signal is produced by PtCuCo-TAs catalyzing luminolH2 O2 system. The amount of miRNA-21 is detected with CL signal. This CL platform performs with limit of detection 0.167 fM and has good selectivity over other RNA. © 2020 Elsevier B.V. All rights reserved.

1. Introduction Noble metals, such as gold (Au), silver (Ag), palladium (Pd) and platinum (Pt) have been applied in oxygen reduction reaction catalysts [1], surface-enhanced Raman spectroscopy [2], fuel cells catalysts [3] and sensors [4] due to their unique catalytic and biocompatible properties. Pt nanomaterial, a typical noble-metal, has been extensively investigated in the application of methanol oxidation reaction [5] and hydrogen oxidation reaction [6] of fuel cells. Although Pt has been considered as a relatively common and effective nanomaterial in the field of catalysis, the sky-rocketing price, limited supply and low durability impedes its widespread practical application [7]. To reduce the cost and maintain the stability of Pt catalyst, many strategies have been used to explore novel Pt catalytic materi-

∗ Corresponding author. E-mail address: [email protected] (X. Hun). https://doi.org/10.1016/j.jpba.2020.113280 0731-7085/© 2020 Elsevier B.V. All rights reserved.

als with low Pt contents. The one commonly adopted strategy is adjusted the morphologies of Pt catalyst which including nanotubes [8], nanoshells [9], nanoparticles [10], nanoclusters [11] et al. For example, nanotubes modified with Pt nanoparticles [12] or Pt monolayer [13] can decrease the amount of Pt effectively, which improving the utilization of Pt and obtaining a high catalytic activity. And the other strategies are introduced a second or third low-cost nonprecious metals to synthesize Pt-based multimetallic alloys. Compared with their monometallic components, Pt-based multimetallic alloys exhibit low consumption, high stability and catalytic activity simultaneously because of synergistic and electronic coordination effect [14]. At present, a great many of Pt-based alloys have been fabricated and extensively explored. For example, Pt-based alloys, including Pt-Ni [15], Pt-Co [16], Pt-Cu [17], Pt-FeCo [18], Pt-Ni-Co [19] and Pt-Cu-Co-Ni [20] and so on, have been proved to be promising alternatives to pure Pt catalysts. Impressively, due to their unique properties, Pt-based alloys have been considered as a promising alternative to pure Pt catalyst for enhancing the catalytic properties especially.

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Benefiting from their unique simple operation, low background, wide dynamic range, fast response, high sensitivity and no need of light excitation, chemiluminescence (CL) as a valuable analytical tool has been attracted considerable attention and widely used in modern analytical chemistry especially bioassays [21–25]. Despite its superiority, luminol-H2 O2 as a typically CL system usually demands the enzymes to enhance the CL efficiency. Therefore, extensive efforts have been dedicated to explore appropriate enzymes, including horseradish peroxidase, peroxidase mimetics and nanozyme for luminol-H2 O2 CL system [26,27]. Compared with those native enzymes, nanozyme catalyzer has not only intrinsic peroxidase-like activity but also higher sensitivity and stability for enhancing luminol-H2 O2 CL system [28,29]. Because the abnormal expression levels of microRNAs (miRNAs) often foreshadows the occurrence of disease, miRNAs act as critical effects in early clinical diagnosis and cancer therapy [30,31]. Therefore, it is significant to detect miRNAs in early prediction. However, it is still a challenge for the detection of miRNAs sensitively and accurately because of short size and low abundance in cells and serum [23,32,33]. For example, the miRNA-21 is at very low concentrations (∼fM to nM) in serum samples [34]. Up to now, various signal amplification methods have been developed to detect miRNAs, including rolling circle amplification [35], cyclic enzymatic amplification method [36] and bicyclic cascade signal amplification [37] et al. In most cases, these signal amplification methods are involved enzyme to assist. Compared with the enzyme-assisted approach, the method of enzyme-free possesses unique advantages containing simple and cheap feature and the elimination of complex enzyme reaction in signal amplification. Therefore, researchers have aimed to design different walker with enzyme-free approaches for signal amplification at the gene level, and it provided an avenue for the development of gene detection [38–40]. For example, two-legged walker had been reported. However, the catalyst dissociation increased when the substrate of microparticle combining with two-legged walker was exhausted. To overcome this difficulty, a simpler single-legged walker with a “cleat” had been designed to prevent the walker dissociating and ensure the walker moving [41,42]. Inspired by the above ideas, a simple, facile, efficient one-step synthesis strategy was used to fabricate PtCuCo trimetallic alloys (PtCuCo-TAs). The Co and Cu as an introduced nonprecious metals with excellent catalytic property were to fabricate Pt-based multimetal alloys. Pt, Cu, Co metal nanomaterials were also respectively synthesized. PtCuCo-TAs gave the best catalyzing for luminol-H2 O2 system. A CL assay based on PtCuCo-TAs-luminol-H2 O2 CL system was fabricated for miRNAs detection coupling single-legged walker strategy. To our best knowledge, this is the first time that the PtCuCo-TAs was applied for catalyzing luminol-H2 O2 CL system for miRNAs detection. 2. Experimental section 2.1. Chemicals and reagents Potassium chloroplatinate (K2 PtCl6 ) was provided by SigmaAldrich. Cobalt (II) chloride hexahydrate (CoCl2 ·6H2 O) and Copper (II) chloride dihydrate (CuCl2 ·2H2 O) were obtained from J&K Chemical Co., Ltd (Beijing, China). Sodium borohydride (NaBH4 ) and ascorbic acid were bought from Aladdin Chemistry Co., Ltd (Beijing, China). Tris-(2-carboxyethyl)-phosphine (TCEP) and Luminol (C8 H7 N3 O2 ) were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Streptavidin-coated magnetic beads (SAMBs) were obtained from Tianjin Baseline (Tianjin-, China). The solutions used in this work were as follows. 10 mM stock solution of luminol was prepared by dissolving luminol in 0.1 M NaOH solution and then stored in the dark at 4 ◦ C. A working solu-

tion of H2 O2 was prepared from fresh 30 % H2 O2 . 10 mM Tris−HCl (pH 8.0) washing buffer containing 0.02 % Tween-20 was prepared to wash SA-MBs. 10 mM Tris−HCl (pH 8.0) reaction buffer with 15 mM KCl and 5 mM MgCl2 was employed to prepare the oligonucleotide solutions. The oligonucleotides were synthesized by Shanghai Sangon Biotechnology Co., Ltd (Shanghai, China) and their sequences of oligonucleotides were list as follows: H1 (Hairpin DNA1): 5 -Biotin-CTTTAGCC GCCATTTC TT TCAACA TCAGTCTG ATAAGCTA CCATGTGTAGA TAGCTT ATCAGACT CCTTGTCA TAGAGCAC-3 miRNA-21 (target): 5 -UAG CUU AUC AGA CUG AUG UUG A-3 H2 (Hairpin DNA2): 5 -ATAAGCTA TCTACACATGG TAGCTT ATCAGACT CCATGTGTAGA G ATGTTGAAA-3 Probe DNA: 5 -SH-CACGAGAT ACTGTTCC TCAGACTA-3 cDNA (Complementary strand of probe DNA): 5 GGAACAGTATCTCGTG-3 Single-base mismatched target RNA (smiRNA-21): 5 -UAG CUU AAC AGA CUG AUG UUG A-3 Two-base-mismatched target RNA (dmiRNA-21): 5 -UAG CAU AUC AGA CUG ACG UUG A-3 Three-base mismatched target RNA (tmiRNA-21): 5 -UAG CAU AUC AGA CCG AUG UUA A-3 miRNA-141: 5 -UAA CAC UGU CUG GUA AAG AUG G-3 miRNA-429: 5 -UAA UAC UGU CUG GUA AAA CCG U-3 let-7a: 5 -UGA GGU AGU AGG UUG UAU AGU U-3 2.2. Apparatus and measurements Transmission electron microscopy (TEM) images were obtained by a JEM-2100 transmission electron microscope instrument. Scanning electron microscopy (SEM) images were recorded by a JSM-6500F machine. X-ray photoelectron spectroscopy (XPS) measurements were taken on an ESCALAB 250Xi spectrometer with an Al monochromatic source. CL signal was collected by RFL-1 CL instrument equipped with a photomultiplier operated at 350 V. Ultrapure water was obtained by Shanghai Yarong Biochemistry Instrument purification system. 2.3. Preparation of PtCuCo-TAs PtCuCo-TAs were synthesized by a simple and rapid reduction method. In briefly, 1.0 mL of metal precursor aqueous solution containing 0.025 M K2 PtCl6 , 0.025 M CoCl2 ·6H2 O and 0.025 M CuCl2 ·2H2 O was added into 5.0 mL of 0.1 M NaBH4 aqueous solution rapidly under stirring at room temperature. The solution was kept stirring for about 5 min and the color was changed from yellow to colorless. Simultaneously, the black products were obtained. As a comparison, the same procedure was used to synthesize pure Pt, Cu and Co metal nanomaterials via adjusting the metal precursors. The binary alloys PtCu, PtCo and CuCo were also synthesized. Finally, the nanomaterials were washed with ultrapure water three times, followed by freeze-dried. The final products were dispersed into ultrapure water and stored at 4 ◦ C for further use. Before using they were sonicated 5 min to obtain 5 mg/mL solution. 2.4. Preparation of the probe DNA modified PtCuCo-TAs and the complex of cpDNA-PtCuCo-TA The probe DNA modified PtCuCo-TA, pDNA-PtCuCo-TA, was synthesized according to previous report with minor revision [43]. Briefly, 100 ␮L 1.0 × 10−7 M probe DNA was activated by 5 ␮L 10 mM TCEP under constant shaking for 1 h at 37 ◦ C in dark conditions. The probe DNA was activated. Subsequently, 100 ␮L PtCuCo-TAs solution was added into and incubated at 37 ◦ C for 24 h under constant shaking. After that, pDNA-PtCuCo-TAs were

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Scheme 1. Scheme of the smart miRNA walking machine based on single-legged walker with a “cleat” for the sensitive detection of miRNA-21.

collected by centrifugation and washed with washing buffer three times. The resulting complex, pDNA-PtCuCo-TA, was dispersed in 200 ␮L reaction buffer. Then 100 ␮L 2.0 × 10−7 M cDNA was added and the mixture solution was incubated at 37 ◦ C for 1 h followed by centrifugation forming the complex (P), defined as cpDNA-PtCuCoTA. 2.5. Synthesis of H1 conjugated MBs 100 ␮L 2 mg mL−1 SA-MBs were washed three times with 100 ␮L washing buffer through the magnetic separation for 5 min. Then the SA-MBs were redispersed in 100 ␮L reaction buffer. Meanwhile, the hairpin DNA, H1 and H2, were obtained by annealing. In brief, 100 ␮L 1.0 × 10-8 M H1 and 100 ␮L 1.0 × 10-8 M H2 were heated at 95 ◦ C for 5 min and cooled to room temperature gradually at a constant rate to form stem-loop structure. Subsequently, 100 ␮L H1 was added into SA-MBs solution and incubated on a shaker at 37 ◦ C for 30 min, followed by washing with washing buffer to remove excess H1. Finally, the resulting H1-MB was dispersed in 200 ␮L reaction buffer.

2.7. Chemiluminescence test The CL detection was conducted on RFL-1 CL instrument. For a typical CL test, 50 ␮L the above dispersion and 50 ␮L H2 O2 at a certain concentration were added into 96-well microplate. Then the CL reaction was triggered by injecting 100 ␮L luminol solution. The quantitative analysis of target miRNA-21 was realized by collecting the CL intensity using analysis software. 2.8. Sample preparation Human serum samples were obtained from Qingdao Central Hospital (Qingdao, China. Ethical Committee License Number: QDCH 2018−05) and were diluted with 0.1 M PB (pH 7.0). 100 ␮L serum solution was treated with 0.2 U/␮L RNase inhibitors. And it was filtrated with 30 K ultrafiltration centrifuge tube. For control experiment, the concentrations were also detected by qRT-PCR [44]. 3. Results and discussion 3.1. Principle of the assay for the detection of miRNA-21

2.6. Procedures for miRNA-21 detection In this work, miRNA-21 was selected as a model target. First, 50 ␮L sample solution contained miRNA-21 and 100 ␮L H1-MB solution were mixed in 1.5 mL centrifuge tube and stored at 37 ◦ C for 2 h to form the complex, miRNA-21/H1-MB (M1). After washing with the washing buffer three times, M1 was dispersed in 200 ␮L reaction buffer subsequently, and 100 ␮L 1 × 10−7 M H2 was added into M1 solution under gentle shaking for 1 h at 37 ◦ C to generate H2/miRNA-21/H1-MB (M2). Then 100 ␮L cpDNA-PtCuCo-TAs solution was added. After reaction 1 h at 37 ◦ C under gentle shaking, cpDNA-PtCuCo-TA/H2/miRNA-21/H1-MB (M3) was gained. After centrifugation, the precipitation was dispersed in 200 ␮L reaction buffer and the dispersion was used for the CL measurement.

The principle of miRNA-21 detection was shown in Scheme 1. In this assay, H1-MB was prepared firstly. In the presence of the walker of miRNA-21, the toehold domain (black and red part) at 3 terminus of miRNA-21 bound to the domain (black and red part) at 5 terminus of H1 to open H1 stem portion and formed M1. Thus, the newly formed ssDNA region (blue and purple part) within H1 was exposed and then interacted with the toehold domain (blue part) of H2 when H2 was added into the solution of M1, forming a tripartite complex M2 consisted of miRNA-21, H1 and H2. In the formation process of M2, the red domains at 3 terminus of H2 and miRNA-21 were both base complementary for the 5 terminus ssDNA of H1, resulting the competition was occurred between H2 and miRNA21. Consequently, the bound red domain of the miRNA-21 can be released easily. Meanwhile, H2 with a bulgy structure was formed.

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Fig. 1. SEM images of Pt (A), Cu (B), Co (C) nanomaterials and PtCuCo-TAs (D).

Due to the existence of “cleat” in the end of miRNA-21 (black part), miRNA-21 hybridized to H1 with a fewer base complementary to prevent miRNA-21 dissociating from H1. When cpDNA-PtCuCo-TAs was added, the ssDNA region (blue part) of H1 reacted with the toehold domain of probe DNA and cDNA was released. Meanwhile, pDNA-PtCuCo-TAs was bound to H1 forming the complex M3. As we all know- that RNAs and DNAs were flexible molecules and can moved in solution [45]. Due to the swing of RNAs and DNAs on the surface of MB, the released red domain of miRNA-21 searched and reacted with the adjacent red domain on the H1 substrates. Because of the branch migration reactions, another H1 was opened, resulting another pDNA-PtCuCo-TAs being captured. With this process repeatedly, a large amounts of pDNA-PtCuCo-TAs was captured onto M3. After separation and washing, the precipitate and H2 O2 were added into the 96-well and luminol solution was injected. The CL signal was produced by PtCuCo-TAs effectively catalyzing luminol-H2 O2 system. The amount of miRNA-21 was quantified by measuring the CL peak intensity.

3.2. Characterization of PtCuCo-TAs, PtCu, PtCo,CuCo, Pt, Cu and Co metal nanomaterials First, the metal nanomaterials of Pt, Cu, Co and the TAs of PtCuCo were prepared via a simple and rapid aqueous solution method. The structural features of these nanomaterials were characterized by SEM. Note that the obtained pure Pt nanoparticles had 3D networklike porous nanostructures, as shown in Fig. 1A. And the diameters of Pt nanoparticles were ranging from 8 to 12 nm. Compared with the 3D network-like porous nanostructures of Pt, the pure Cu nanomaterials were rough and composed of many 3D irregular spherical

nanoparticles with porous structures and the diameters of Cu nanomaterials were about 45 ± 3 nm as shown in Fig. 1B. Furthermore, Fig. 1C exhibited the morphology of Co nanomaterials, and it was found that Co nanomaterials displayed uniform flower-like nanostructures with diameter up to 160 ± 10 nm. Simultaneously, the images of Fig. 1D revealed that PtCuCo-TAs had irregular bulk structure. The results of morphology for different samples revealed that PtCuCo-TAs was synthesized successfully. It was found that binary alloys PtCu, PtCo gave relative lower CL signal than that of CuCo [46]. And PtCuCo-TAs gave higher CL signal than that of CuCo. So the characterizations of binary alloys were not performed. Because PtCuCo-TAs gave the strongest CL, the composition of PtCuCo-TAs was further studied. XPS measurements were carried out to give detailed analysis of the chemical composition and the element state of PtCuCo-TAs. As shown in Fig. 2A, the XPS spectrums of PtCuCo-TAs were dominated by Pt, Cu and Co elements, revealing that the metal salt aqueous solution was successful reduced distinctly. The obtained Pt 4f spectrum was shown in Fig. 2B. And the Pt 4f spectrum exhibited two strong peaks at binding energies of 71.3 and 74.5 eV which corresponding to 4f5/2 and 4f7/2 of Pt, respectively. According to the results, it could be seen that these two peaks were divided into two pair of peaks at around 71.2 eV, 74.6 eV and 73.0 eV, 76.8 eV, respectively. The results revealed that Pt had two valence states: the one was zero-valent metal phase of Pt (71.2 eV and 74.6 eV), and the other was platinum oxide species or Pt(OH)2 (73.0 eV an 76.8 eV) [20,47,48]. Compared with the relative intensities of another phase of Pt, it could be concluded that zerovalent metal phase of Pt was dominated among the composition. The Cu 2p XPS spectrum of PtCuCo-TAs was exhibited in Fig. 2C. Apparently, the Cu 2p3/2 peaks appeared at 932.7 eV and the Cu

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Fig. 2. XPS spectrum of PtCuCo-TAs (A). And XPS spectra of Pt 4f (B), Cu 2p (C) and Co 2p (D) regions for PtCuCo-TAs, respectively.

2p1/2 peaks appeared at 952.4 eV [49,50] indicating the existence of zero-valent metal phase of Cu. Likewise, in Fig. 2D deconvolution of the Co 2p region displayed peaks at 782.5 and 785 eV, and these peaks were assigned to zero-valent metal phase of Co and cobalt oxide, respectively [48]. On the basis of these XPS measurements, it demonstrated that the metal salt aqueous solution was reduced successfully. 3.3. Chemiluminescence behavior of the Pt, Cu, Co nanomaterials and the PtCuCo-TAs to catalyze luminol-H2 O2 CL system The catalytic activities of Pt, Cu, Co nanomaterials and PtCuCo-TAs for the luminol-H2 O2 CL system were studied. The nanomaterials solutions were prepared by dispersing the nanomaterials into water and ultrasonic treatment for 10 min. Subsequently, H2 O2 solution was mixed with different above products aqueous solution and then luminol was injected. As shown in Fig. 3A, without nanomaterials, no obvious CL emission was observed (blank signal) (cure a). Compared with the blank signal, the nanomaterials of Pt, Cu and Co showed relatively strong CL activity (curve b, c, d). For the PtCuCo-TAs, a strongest CL emission was observed (curve e). Compared to the CL emission in the presence of nanomaterials, it revealed that the excellent catalytic performance of PtCuCo-TAs toward luminol-H2 O2 CL system. The high CL intensity is due to the merit of high catalytic efficiency of nanozyme catalyzer. Therefore, PtCuCo-TAs was selected as excellent catalyst for further experiment. In order to better understand Pt, Cu, Co nanomaterials and PtCuCo-TAs catalyzing luminol-H2 O2 CL system, the CL kinetic behaviors were studied (Fig. 3B). After luminol being injected, the luminol-H2 O2 CL signal reached a maximum value of 113 within 0.5 s in the absence of nanomaterials. And then the CL signal decreased quickly (curve d). When Cu nanomaterials were mixed with H2 O2 , luminol was injected; a relatively enhanced CL emission

was recorded. The CL intensity was reached to 441 (curve e). At the same time, when luminol was injected into Pt-H2 O2 , Co-H2 O2 and PtCuCo-TAs-H2 O2 , respectively, the kinetic curves were observed and rapid increase to maximum values of 791 (curve c), 1945 (curve b) and 5520 (curve a), respectively. In comparison, the PtCuCo-TAs had outstanding CL catalytic performance for luminol-H2 O2 CL system. And the luminol-H2 O2 CL signal enhanced by PtCuCo-TAs was more about 3 times stronger than other nanomaterials. Therefore, PtCuCo-TAs was the optimal nanomaterial for the luminol-H2 O2 CL system. 3.4. Optimization of chemiluminescence measurement conditions According to the CL mechanism of luminol-H2 O2 , it requires optimal conditions to produce stronger light emission. Therefore, the effect of the H2 O2 concentration was investigated as shown in Fig. 4A and B. And Fig. 4A and B depicted the background CL intensity of I0 (black curves) that was obtained in the absence of PtCuCo-TAs and the CL signal of I (blue curves) was gained in the presence of PtCuCo-TAs with a series of H2 O2 concentration, respectively. The results indicated that the I and I (I0 –I) sharply increased with the concentration of H2 O2 ranging from 0.1–5 mM and then slowly decreased with the increasing of the H2 O2 concentration from 5 to 30 mM. Consequently, the H2 O2 concentration at 5 mM was selected for this luminol-H2 O2 CL system. Fig. 4C and D described the effect of the pH on CL intensity in the presence of 5 mM H2 O2 . It could be seen that the CL signal of I increased with an increasing of pH from 8.0–9.5 and above which it decreased. Meanwhile, I0 also increased with the pH value ranging from 8.0–8.5. However, I0 sharply decreased with the pH value increasing from 9 to 11 and then increased with the pH value increasing. So, I was used to evaluate the effect of pH. As shown in Fig. 4D, the I was increased with the pH value to 9.5 and then decreased with the pH value ranging from 10.0–12.0. Therefore, the

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Fig. 3. (A) CL features of luminol-H2 O2 system catalyzed by different nanomaterials. blank (ultrapure water was used as the blank displaced the nanomaterials) (a), Pt (b), Cu (c), Co (d) and PtCuCo-TAs (e). (B) CL kinetic curves of luminol-H2 O2 system catalyzed by PtCuCo TAs (a), Co nanomaterial (b), Pt nanomaterial (c), blank (ultrapure water was used as the blank displaced the nanomaterials) (d) and Cu nanomaterial (e). Insert: magnification of curve b, curve c, curve d. (C) CL spectra of the luminol-H2 O2 -PtCuCo-TAs (a), luminol-H2 O2 -Co (b), luminol-H2 O2 -Pt (c), luminol-H2 O2 -Cu (d), luminol-H2 O2 (e) and luminol-dissolved oxygen (f). Insert: magnification of curves (e) and (f). (D) CL intensity of luminol-H2 O2 -PtCuCo-TAs system in the presence (a) and absence (b) of dissolved oxygen, in the presence of ethanol (c) and ascorbic acid (d), respectively. H2 O2 : 5 mM; Luminol: 1 mM; pH: 9.0; ascorbic acid: 1 mM; ethanol: 1 mM.

pH value at 9.5 was selected as the optimal pH value in the following studies. Comparing with most common luminol-H2 O2 CL system under strongly alkaline condition, this luminol-H2 O2 -PtCuCo-TAs CL system emitted strong CL under alkalescent conditions [27]. Therefore, this CL system can be used to develop novel CL assays under close to physiological conditions. As shown in Fig. 4E and F, the influence of the luminol concentration was studied. It was found that I0 and I were increased with the concentration of luminol increasing from 0.01 to 1 mM and then remained almost stable. At the same time, I was increased with the concentration of luminol increasing from 0.01 to 1 mM and then remained almost stable with the increasing of luminol concentration ranging from 5 to 10 mM. Considering this result, 1 mM luminol was adopted for this experiment. 3.5. Sensitivity of miRNA-21 detection Therefore, the single-legged walker with a “cleat” enzyme-free CHA CL method was used for sensitive detection of miRNA-21. Under optimal experimental conditions, the CL signal with various concentrations of target miRNA-21 was investigated. As shown in Fig. 5A, the CL intensity was enhanced gradually with the increasing of target miRNA-21 concentration. Meanwhile, in Fig. 5B, the CL signal was displayed to be a favorable logarithmically relating to the concentration of target miRNA-21 in the range of 0.5 fM to 100.0 pM. The linear regression equation obtained was I = 1496.70 logCmiRNA-21 + 23967.64 with a correlation coefficient (R2 ) of 0.9974. The reproducibility was explored by five parallel measurements of 5.0 pM target miRNA-21 with the relative standard deviation (RSD) values of 4.3 %, and the detection limit of this

method was 0.167 fM at a signal-to-noise ratio of 3 (S/N = 3). In addition, compared with other reported methods, the analytical performances of various methods were listed in Table 1 and this method showed a better performance. The LOD of 0.167 fM was lower than other most reported methods (Table 1). Therefore, it is a capable assay to detect target miRNA-21. 3.6. Selectivity The selectivity test was further carried out to detect miRNA21 on this assay. miRNA-21, smiRNA-21, dmiRNA-21, tmiRNA-21, miRNA-141, miRNA-429, let-7a and the mixture RNAs solution of both miRNA-21 and other six miRNAs were selected to study the selectivity under the optimal conditions (The concentration of miRNA-21 and other six miRNAs were 5 × 10−15 M and 5 × 10-14 M, respectively.). As shown in Fig. 6, no obvious CL responses were detected of other six miRNAs. However, the CL response of miRNA21 which 10-fold concentration lower than other miRNAs displayed the highest among other six miRNA obviously. Furthermore, the CL signal of the mixture RNAs solution was slightly higher than the CL signal of miRNA-21. Comparing with the CL signal of miRNA21, the CL response of other kinds of miRNAs was negligible in reaction buffer. In order to explore the specificity of this biosensor in health human serum, further experiments were carried out in human serum samples and the CL signal was recorded. As shown in Fig. 6, the health human serum had no obvious CL response and the human serum with 5 × 10−15 M miRNA-21 was slightly lower than the CL signal of miRNA-21 in reaction buffer. These results indicated that the serum had no significant effect for the detection of miRNA. The results well illustrated that this assay had satisfac-

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Fig. 4. The effect of H2 O2 concentration (A, B), pH (C, D) and luminol concentration (E, F) on CL intensity. CL curves I (blue curves, left) and I0 (black curves, right) represent the CL intensity in the presence and absence of PtCuCo-TAs, respectively, and I = I - I0 . H2 O2 : 0.1, 0.6, 1, 5, 10, 20, 30 mM; pH: 8, 8.5, 9, 9.5, 10, 10.5, 11.0, 11.5, 12.0; Luminol: 0.01, 0.05, 0.1, 0.5, 1, 5, 10 mM (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article).

Fig. 5. (A) CL responses of miRNA-21. (a-h) 0, 0.5 fM, 1.0 fM, 10.0 fM, 100.0 fM, 1.0 pM, 10.0 pM, 100.0 pM. (B) Calibration plot of the logarithmic concentrations of target miRNA-21 vs CL intensity. pH: 9.5; H2 O2 : 5 mM; Luminol: 1 mM.

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Table 1 Performance of various methods for the detection of miRNA-21. Methoda

Materials

Detection range (M)

Detection limit (M)

Ref

PEC FRET DPV DPV Flu CL

TiO2 /Au/ZCIS/N-CDs ssDNA-MoS2 -PEG-FA FcDNPs UCNPs@DNA “DD − A” FRET DNA-PtCuCo TAs

1 × 10−12 - 1 × 10-7 1 × 10−8 - 5 × 10−8 1 × 10−12 - 1 × 10-8 1 × 10−16 - 5 × 10-13 1 × 10−10 - 4 × 10-9 5 × 10−16 - 1 × 10-10

3.1 × 10−11 -b 3.1 × 10−14 4.1 × 10−17 3.4 × 10−11 1.67 × 10−16

[51] [52] [53] [54] [44] c

c

This work. Not reported. a PEC, Photoelectrochemical; FRET, Fluorescence resonance energy; DPV, Differential pulse voltammetry; Flu, Fluorescence; ZCIS, CuInS2 /ZnS quantum dots; N-CDs, Ndoped carbon dots; ssDNA-MoS2 -PEG-FA, Folate-functionalized MoS2 nanosheets immobilized with fluorescence-labeled ssDNA probes; FcDNPs, ferrocene-labeled bipedal DNPs; UCNPs@DNA, Upconversion nanoparticles combined with DNA; “DD − A” FRET, Donor donor − acceptor fluorescence resonance energy transfer. b−

Table 2 The determination of miRNA-21 in serum samples (n = 7). Samplea

1 2 3 4 5 a

Found (nM)

Added (nM)

This method

Control method

0.0278 0.0295 0.0231 23.6 17.5

0.265 0.0298 0.0240 23.4 17.4

0.02 0.05 0.1 20.0 10.0

Detected (nM) This method

Control method

0.0482 0.0817 0.1207 43.7 27.4

0.0480 0.0789 0.1238 43.8 27.4

Recovery ratio (%)

RSD (%)

102.0 104.4 97.6 100.5 99.0

3.6 4.3 3.7 2.8 3.1

1, 2, 3: serum sample from healthy people; 4, 5: serum sample from breast cancer patient.

apply for the detection of miRNA-21 with accuracy and reliability in sample analysis. 3.8. Possible chemiluminescence mechanism

Fig. 6. Selectivity investigations of this assay for detection of miRNA-21. a to h: blank, miRNA-21, smiRNA-21, dmiRNA-21, tmiRNA-21, miRNA-141, miRNA-429, let-7a; i: the mixture miRNAs solution of miRNA-21 and other six miRNAs; j: human serum; k: the mixture solution of miRNA-21 and human serum; miRNA-21 concentration: 5.0 × 10−15 M; other six miRNA concentration: 5.0 × 10-14 M; pH, 9.5; H2 O2 , 5 mM; Luminol, 1 mM.

tory selectivity for the detection of miRNA-21. Hence, this assay can detect miRNA-21 with outstanding selectivity. 3.7. The detection of miRNA-21 in serums To validate the applicability of this method for the detection of miRNA-21, further experiments were implemented in human serum. Various concentrations of miRNA-21 were added into the health human serum and breast cancer patient serum. After seven parallel detection processes, the results was demonstrated in Table 2. The recovery rates of miRNA-21 vary from 99.0–104.4% in the serum samples and the relative standard deviation was less than 4.3 % (n = 7), revealing the excellent consistency with the added concentrations. The precision and accuracy data of withinand between-days were also studied (Table S1). The concentration of miRNA-21 in health human serum is lower than that in the breast cancer patient serum. The results were in good accordance with control method [44,55,56]. Hence, this method can effectively

In order to investigate the CL mechanism of different nanomaterials to catalyze luminol-H2 O2 CL system, the CL spectra of the Pt, Cu, Co nanomaterials and PtCuCo-TAs for catalyzing the luminolH2 O2 CL system were further expected to investigate. Therefore, the CL spectra were studied by a fluorospectrometer which was depicted in Fig. 3C. The experiments demonstrated that luminol (cure f) and luminol-H2 O2 (cure e) had weak CL intensity and generated low CL signal. Luminol-H2 O2 -Co (cure b), luminol-H2 O2 -Pt (cure c), and luminol-H2 O2 -Cu (cure d) generated strong CL emission. Meanwhile, the results clearly testified that the PtCuCo-TAs gives the strongest CL intensity (cure a). More importantly, the maximum emission wavelength of luminol CL system centered at 425 nm either in the absence or in the presence of the nanomaterials. It was revealed that the excited-state 3-aminophthalate anions (3-APA*) were still the luminophore in these luminol CL systems and the introduction of nanomaterials did not generate the new emitting species [57,58]. The mechanism of luminol-H2 O2 -PtCuCoTAs was studied systematically. To explore the mechanism of the CL system of luminol-H2 O2 PtCuCo-TAs, the effects of dissolving oxygen and reactive oxygen • species (ROS) including superoxide radical anion (O2 − ), and • hydroxyl radical ( OH) were further investigated. Therefore, nitrogen and active oxygen radical scavengers were introduced to identify the intermediates of the CL reaction. As shown in Fig. 3D, the CL intensity was sharply decreased in nitrogen-saturated solution (cure a) comparing with the CL intensity in air-saturated solution (cure b), indicating that the dissolved oxygen was catalyzed by PtCuCo-TAs to generate superoxide anions for enhancing CL signal. This result revealed that the dissolved oxygen was participated in this CL system to some extent. Furthermore, ethanol and • ascorbic acid, two well-known effective radical scavengers for O2 − , • and OH [59,60], respectively, were introduced for verifying the presence of these ROS. The influence of ethanol and ascorbic acid on the CL intensity of this system was shown in Fig. 3D, and the great quench (cure c, d) of CL intensity has been observed in the presence

S. Mei, B. Liu, X. Xiong et al. / Journal of Pharmaceutical and Biomedical Analysis 186 (2020) 113280

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Scheme 2. Schematic illustration of the CL mechanism for the luminol-H2 O2 -PtCuCo-TAs system.

of ethanol and ascorbic acid. Comparing with the quenching ability of ethanol (cure c), ascorbic acid was more significantly quenches in the CL signal (cure d). These results confirmed that the generation of ROS played a key role in the luminol-H2 O2 -PtCuCo-TAs CL emission process. Based on the above experiments, the possible CL mechanism of luminol-H2 O2 -PtCuCo-TAs system was concluded and shown in Scheme 2. Owing to the synergetic catalytic activity of PtCuCo-TAs, the H2 O2 and dissolved oxygen would first rapidly decomposed • • • to generate a great number of OH and O2 − . However, O2 − was unstable in aqueous solution because it would initiated an active oxygen chain reaction, forming a single oxygen (1 O2 ) (process A). Meanwhile, luminol and H2 O2 decompose to produce luminol− and HO2 − , respectively (process B). Subsequently, the as-formed • luminol− and HO2 − would react with OH to accelerate the forma•− • tion of luminol . Then, the formation of luminol − reacted with •− 1 both O2 and O2 , generating an unstable 3-APA* which would quickly returned to the ground-state with the strong light emission at 425 nm (process C). These results were consistent with the results of CL spectral (Fig. 3 C). And the detailed chemical reaction equation was provided in Supporting Information (Fig. S1).

amplifying detection signal with enzyme-free. Furthermore, the assay benefits the advantages of facile, specificity and high sensitivity, and excellently discriminative ability for the detection of miRNA-21. By altering the recognition substances, the assay could be extended to detect other miRNA as well as DNA in practice. Hence, we conceive that the single-legged walker triggering and nanozyme amplifying assay has widespread application in versatile detection. CRediT authorship contribution statement Shuyu Mei: Conceptualization, Methodology, Writing - review & editing, Formal analysis, Funding acquisition. Bingru Liu: Methodology, Data curation, Writing - original draft, Conceptualization. Xiaoli Xiong: Visualization, Investigation. Xu Hun: Supervision, Validation, Writing - review & editing, Project administration, Funding acquisition. Declaration of Competing Interest The authors declared that they have no conflicts of interest to this work.

4. Conclusions Acknowledgments In summary, we have constructed a single-legged walker with a “cleat” on the surface of MB using miRNA-21 as trigger, which was used as an effective assay to sensitively detect target miRNA21. Furthermore, a facile and fast method was used to synthesize PtCuCo-TAs acting as nanozyme, which had not only intrinsic peroxidase-like activity but also higher stability for enhancing luminol-H2 O2 CL system. By taking advantage of both the singlelegged walker and the PtCuCo-TAs nanozyme, a single-legged walker triggering assay was carried out on the surface of MB and a CL signal amplification was obtained by DNA-PtCuCo-TAs and enemy-free CHA, indicating that this method had an effectively

This work was supported by National Natural Science Foundation of China (21575073, 81372810), Applied Basic Research Plan Project of Datong City, China (2018158), Laoshan Scholar Program of Qingdao University of Science and Technology (201802685), Shanxi Province University Science and Technology Innovation Project (751), Shanxi Province Innovation and Entrepreneurship Training Program for College Students (2019446), National Innovation and Entrepreneurship Training Program for College Students (201910120001), Doctoral Scientific Research Starting Foundation of Shanxi Datong University (2013-B-18).

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