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Dual-modal split-type immunosensor for sensitive detection of microcystin-LR: enzyme-induced photoelectrochemistry and colorimetry Jie Wei, Weidan Chang, Aori Qileng, Weipeng Liu, Yue Zhang, Shiya Rong, Hongtao Lei, and Yingju Liu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02546 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 10, 2018

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Analytical Chemistry

Dual-modal split-type immunosensor for sensitive detection of microcystin-LR: enzyme-induced photoelectrochemistry and colorimetry Jie Wei1,2, Weidan Chang1,2, Aori Qileng1, Weipeng Liu1, Yue Zhang1, Shiya Rong1, Hongtao Lei2, Yingju Liu1,* 1

Department of Applied Chemistry, College of Materials and Energy, South China Agricultural University, Guangzhou 510642, China 2 The Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University, Guangzhou 510642, China *Fax: 86-20-85285026, Phone: 86-20-85280319, E-mail: [email protected] (Y. Liu)

ABSTRACT: Microcystins, the lethal cyanotoxins from microcystis aeruginosa, can inhibit the activity of protein phosphatase and promote liver tumor. Herein, a dual-modal split-type immunosensor was constructed to detect microcystin-LR (MCLR), basing on the photocurrent change of CdS/ZnO hollow nanorod arrays (HNRs) and the blue-shift of surface plasmon resonance peak from Au nanobipyramids@Ag. By using mesoporous silica nanospheres as the carrier to immobilize secondary antibody and DNA primer, hybridization chain reaction was adopted to capture alkaline phosphatase, while its catalytic reaction product, ascorbic acid, exhibited dual functions. The detailed mechanism was investigated, showing that ascorbic acid can not only act as the electronic donor to capture the holes in CdS/ZnO-HNRs, leading to the increase photocurrent; but also as the reductant to form silver shells on Au nanobipyramids, generating multiply vivid color variations and blue shifts. Compared with traditional photoelectrochemical immunosensor or colorimetric method for MC-LR, a more accurate and reliable result can be obtained, due to different mechanism and independent signal transduction. Therefore, this work can not only propose a new dual-modal immunosensor for MC-LR detection but also provide innovative inspiration for constructing sensitive, accurate and visual analysis for toxins. INTRODUCTION

With the increasing of global warming and water pollution, lakes are facing the problem of cyanobacteria eruption. Frequent cyanobacteria outbreak releases a variety of harmful cyanotoxins, which cause damage to drinking water, aquatic animal species and public health. Microcystins (MCs), as the most widely existent cyanotoxins, induce multiple toxic effects, leading to the injury of immune, urinary, digestive or reproductive system.1 Among MCs, common isomers include MC-YR, MC-RR and MC-LR, with various compositions of tyrosine (Y), arginine (R) or leucine (L).2 The World Health Organization (WHO) has assigned 1 µg/L as the provisional guideline for the concentration limit of MC-LR in water. Some analytical techniques, including liquid chromatography-mass spectrometry,3 high-performance liquid chromatography,4 protein phosphatase immunoassay,5 enzyme-linked immunosorbent assay6-8 and aptasensor, 9,10 have been used to detect MC-LR, but the accuracy, sensitivity and convenience are still pursued. Currently, photoelectrochemical (PEC) sensing has been rapidly developed by virtue of excellent analytical performance, low-cost equipment and easy miniaturization. 11-14 To fabricate a typical PEC immunosensor, biological receptors are employed as recognition element. Meanwhile, the light is utilized to the excitation source and the photocurrent is adopted as detection signal. Since the excitation and detection signal belong to different forms of energy, the background noise can be greatly reduced. Most PEC

sensors are usually analyzed based on single signal change caused by the target.15-17 However, the response may be influenced by external interferences including different personnel operating, nonstandard test process or diverse experimental environment. Therefore, the uncertainty may be existed by using the detection from single-modal readout. To meet the accuracy and sensitivity of quantitative analysis, the dual-signal method has been adopted. For instance, Yuan’s group proposed a ratiometric PEC assay by using CdS QDs and SiO2@MB as reduced and enhanced markers at wavelengths of 460 nm and 623 nm, respectively,18 while Wang’s group constructed a dual channel self-reference PEC biosensor with “signal on” and “signal off” models.19 More recently, Wei’s group designed a colorimetric and fluorescence detection of cardiac troponin I (cTnI)20 and Chen’s group accomplished a PEC immunoassay with complementary fluorescent detection.21 Until now, no strategy is constructed from dual-modal strategy by a PEC immunosensor with colorimetric detection. The colorimetric detection has been extensively investigated due to low cost, simplicity and observation with eyes.22-24 However, most of them can be only used for qualitative detection owing to the limited colors. For instance, the chromogenic substrate, 3,3′,5,5′-tetramethylbenzidine, can be catalyzed by horseradish peroxidase or peroxidaselike nanomaterials, showing monochromic intensity change in the presence of H2O2.25,26 Human eyes are not sensitive

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to the intensity change of the same color. It is necessary to develop a multicolor immunoassay for the naked-eye observation. Noble metal nanoparticles are potential candidates owing to their tunable morphology and localized surface plasmon resonance (LSPR) peak.27,28 Nevertheless, conventional colorimetric biosensors from the aggregation of metal nanoparticles are vulnerable to impurities or ionic strength, exhibiting relatively low sensitivity.29,30 In contrast, in-situ synthesis of bimetallic nanoparticles by enzyme-induced silver deposition can regulate the shape, size, composition of product, while showing a series of vivid color variations to improve the sensitivity without the background. Unlike the round end-shape of Au nanorods or Au nanoparticles, Au nanobipyramids (Au NBPs) with sharper tips show a higher sensitivity to refractive index.31 Therefore, both local electromagnetic field enhancement and the extinction cross section of Au NBPs are higher, making them more favorable for plasmon enhanced spectroscopy.32,33 In this work, a dual-modal split-type PEC immunosensor with complementary colorimetric detection for MC-LR in water was tailored. In order to achieve this design, CdS/ZnO hollow nanorod arrays (HNRs) with excellent photoelectric properties were directly grown on Fdoped SnO2 (FTO) as the photoelectrode. Then, the immune reaction was carried out in the microplate, while hybridization chain reaction (HCR) triggered formation of enzymatic concatamer on mesoporous silica nanospheres (MSNs) to achieve signal amplification. The enzymatic hydrolysate, ascorbic acid (AA), can act as an electron donor to capture the holes generated from CdS/ZnO-HNRs, which can produce a “signal-on” PEC detection. In addition, AA can induce in-situ silver (Ag) metallization on Au NBPs to form Au NBPs@Ag, resulting a series of vivid color variations and blue shift of the LSPR band. Benefiting from different mechanism and relatively independent signal transduction, the interference between two signals in the dual-modal immunosensor can be greatly reduced. EXPERIMENTAL SECTION

Preparation of CdS/ZnO-HNRs on FTO. Vertically aligned ZnO nanorod arrays (NRs) were directly grown on FTO. Firstly, by dissolving 0.1098 g Zn(Ac)2 in 100 mL ethanol, ZnO seed precursor was produced and then spun onto the FTO surface at 6000 r/min. After the FTO was calcined in muffle furnace at 320 °C for 60 min, it was placed with the conductive surface facing in the growth solution of 40 mM methenamine and 40 mM zinc nitrate. After heating at 95 °C for 5 h and washing, the ZnO NRs/FTO was obtained. Secondly, CdS nanoparticles were decorated on the ZnO NRs/FTO by immersing in a solution containing 10 mM cadmium nitrate and 10 mM thioacetamide. After heating at 40 °C for 30 min and washing, it was annealed in tubular furnace at 500 °C (rate: 3 °C/min) for 1 h to form the CdS/ZnO-HNRs. Preparation of Ab2-MSNs-S0 conjugate. Firstly, mesoporous silica nanospheres (MSNs) were prepared as follows.

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Typically, 0.1 g hexadecyl trimethyl ammonium bromide (CTAB) was dissolved in 7 mL water, then ethanol (1.8 mL), ether (3 mL), ethylsilicate (TEOS, 1 mL) and ammonia (0.1 mL) were successively injected. After stirring for 4 h, the product was centrifuged, washed and calcined at 550 °C. Then, MSNs were aminated by dispersing 10 mg of MSN in 7 mL of ethanol and treating with 0.1 mL of (3aminopropyl) triethoxysilane (APTES). After stirring for 24 h at 30 °C, the suspension was collected and scattered in Tris-HAc solution. Secondly, the Ab2-MSNs-S0 conjugate was prepared. After 2 mL of aminated-MSNs was ultrasonicated for 20 min, 5 µL of Ab2 (1 mg/mL) was dropped and blended for 30 min. Immediately, 100 µM S0 (100 µL) was added and gently stirred at 4 °C for 24 h. Therefore, Ab2-MSNs-S0 was prepared by the covalent combination of amino groups of MSNs with carboxyl groups of Ab2 or aldehyde groups of S0. Preparation of Au NBPs. By using seed-mediated-growth technology, Au NBPs were prepared as follows.34 Namely, 0.25 mL of fresh NaBH4 (25 mM) was rapidly injected into 10 mL solution of 50 mM hexadecyl trimethyl ammonium chloride (CTAC), 0.25 mM HAuCl4 and 5 mM citric acid. After stirring vigorously for 2 min, the mixture was heated at 80 ºC for 90 min under gentle stirring, with the color changing from brown to red, suggesting gold seeds have been formed. Then, 10 mL of gold seeds were added into a growth mixture containing HAuCl4 (10 mM, 10 mL), CTAB (100 mM, 200 mL), HCl (1 M, 4 mL), AA (100 mM, 1.6 mL) and AgNO3 (10 mM, 2 mL) at 30 °C for 2 h to produce Au NBPs. The purification of Au NBPs can be found in the supporting information. Fabrication of dual-modal PEC and colorimetric immunosensor. The construction of dual-modal PEC and colorimetric immunosensor was shown in Scheme 1A. (1) Firstly, 50 µL of 1 mg/mL dopamine was dropped into the 96-microwell plate and incubated for 30 min at 37 °C. After drying, MC-LR antigen (10 µg/mL, 20 µL) was added and incubated overnight at 4 °C, followed by adding 20 µL of blocking reagent to block nonspecific sites. According to the competitive method, 20 µL mixture with equal volume of MC-LR antibody (Ab1, 5 µg/mL) and MC-LR with different concentrations were enclosed. (2) After the specific binding of biomolecules, 20 µL of Ab2-MSNs-S0 conjugate was incubated, followed by adding a mixture of S1 (2.5 µM) and S2 (2.5 µM) at 37 °C for 2 h. Subsequently, 20 µL of 15 µg/mL streptavidin-labeled alkaline phosphatase (SAALP) was injected and incubated for 60 min, followed by adding 310 µL of DEA buffer (0.4 M, pH 9.8) containing 100 mM ascorbic acid 2-phosphate (AAP) and incubating at 37 °C for 20 min. (3) Finally, 300 µL of the solution in the well was transferred for PEC measurement; another 10 µL of the solution was transferred into the mixture containing AgNO3 (5 mM, 50 µL) and Au NBPs (2.0 au at 670 nm, 50 µL), followed by incubating for 5 min at 37 °C, terminating the reaction with Na3VO4 (10 mM, 50 µL) and recording the UV-vis absorption spectra between 300 and 800 nm for colorimetric detection.


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Analytical Chemistry c) indicated that CdS, with (100), (002), (101), (110) and (114) peaks at 24.9°, 26.7°, 28.3°, 43.9° and 72.8° (JCPDS 80-0006), was successfully deposited on ZnO NRs. Fig. 1I showed the UV-vis reflection spectra, where approximate 400 nm of the absorption edge for ZnO NRs can be found, suggesting a near ultraviolet adsorption and 3.1 eV for the bandgap (curve a). However, the deposition of CdS on ZnO-HNRs (curve b) can shift the absorption edge to 570 nm, implying 2.2 eV of the bandgap. Therefore, the narrowing of bandgap of ZnO NRs by CdS nanoparticles can improve the light absorption shortage of long wavelength region of pure ZnO NRs. The inset of Fig. 1I was photographs of bare ZnO NRs and CdS/ZnO-HNRs on FTO, showing that the deposition of CdS nanoparticles can cause the color from gray to orange.

Scheme 1. Schematic diagram of the construction (A) and the response mechanism (B) of dual-modal PEC and colorimetric immunosensor.

RESULTS AND DISCUSSION

Characterization of CdS/ZnO-HNRs. Fig. 1A and B showed SEM images of ZnO NRs in different scales, which have ordered shapes with hexagonal apex diameters from 100 nm to 200 nm. In addition, ZnO NRs were vertically oriented on the basement with a high coating density (Fig. 1A). Remarkably, a relatively smooth surface morphology can be found (Fig. 1B). After depositing a layer of CdS nanoparticles, the rod smooth surface of ZnO NRs formed many granular protrusions from the bottom to the top ends of the rods. Thus, significant surface roughening, topographical change and diameter increase can be noted (Fig. 1C and D). In addition, the TEM image (Fig. 1E) provides a confirmation for the hollow nanorod structure, causing by the partly corrosion of ZnO NRs.35 The EDS of CdS/ZnOHNRs was displayed in Fig. 1F, showing that elements including Zn, O, Cd and S can be found. Furthermore, elemental mappings have been investigated (Fig. 1G), which certified that all the elements were uniformly distributed on the surface. X-ray diffraction (XRD) analysis was implemented to discover the crystal structure of the sample. Except for the peaks from FTO (Fig. 1H, curve a), some new characteristic diffraction peaks appeared at 34.4°, 36.3°, 47.5°, 62.9° and 72.6°, indexing to the (002), (101), (102), (103) and (004) peaks of ZnO with a hexagonal structure (JCPDS No. 74-0534) (curve b). The XRD pattern (curve

Fig. 1. SEM image of ZnO NRs(A, B), CdS/ZnO-HNRs (C, D) in different scales, (E) TEM image of CdS/ZnO-HNRs, (F) EDS spectrum of CdS/ZnO-HNRs, (G) Elemental mapping of CdS/ZnO-HNRs, (H) XRD of FTO (a), ZnO NRs (b) and CdS/ZnO-HNRs (c), (I) UV-vis spectra of ZnO NRs (a) and CdS/ZnO-HNRs (b).

Characterization of Ab2-MSNs-S0 conjugate. MSNs were synthesized by the sol-gel method using TEOS as the silicon source, CTAB as the template and ether/ethanol as the cosolvent. Fig. 2A showed that the products were coarse surface and homogeneous morphology. TEM images (Fig. 2B and 2C) revealed that MSNs presented a radially flower structure with good monodispersity and a mean diameter of about 250 nm. Obviously, the large number of narrow and ordered channels on MSNs can provide larger specific surface, which are benefit for the biomolecule immobilization (the morphology can be controlled by adjusting the amount of TEOS, Supporting information). After amination, MSNs can be covalently bonded with carboxylcontaining Ab2 or aldehyde-modified S0, which was described by Circular Dichroism (CD) spectra. In Fig.2D, there was a positive peak at about 275 nm for S0 (curve a), the characteristic absorption of single-strand DNA. In curve b for the spectrum of Ab2, two negative peaks at about 209 and 223 nm appeared, demonstrating that the structure of protein was mainly α-helical secondary struc-



ture.36 As for Ab2-MSNs-S0 conjugate (curve c), the absorption peaks of Ab2 and S0 were both detected, proving that biomolecules were successfully modified on MSNs without changing their secondary structure. To prove the formation of dsDNA after HCR, the gel electrophoresis was investigated. As in Fig.S2, lane 1 was DNA marker and lane 2-4 were images for S0, S1 and S2, while the base number of S0 was too short to escape from the gel. Lane 5 was the image of the mixture containing S1 and S2 after incubating for 2 h at 37°C. It was similar to that of pure S1 and S2, suggesting that HCR cannot be triggered in the absence of S0. However, in the presence of S0, the polymer chain product could be clearly seen (lane 6), indicating the self-assembly of dsDNA with a higher molecular weight through HCR.

Fig. 2. (A) SEM image of MSNs, (B, C) TEM images of MSNs at different scales, (D) CD spectra of (a) S0, (b) Ab2 and (c) Ab2MSNs-S0 conjugate.

Characterization of Au NBPs. Au NBPs were prepared by growing Au seeds in the presence of CTAC and citric acid. The yield of Au NBPs was close to 70%, since Au NBPs and Au nanospheres (NSs) can be both found but it was difficult to separate Au NBPs from Au NSs (Fig.3B). In order to obtain homogeneous Au NBPs, the purification was carried out by using Ag shell growth, depletion forceinduced self-separation and Ag shell etching (illustrated schematically in Fig.3A). Firstly, after the silver shell growing, the product showed the structure of core-shell Au@Ag NSs (Fig.3C) and Au@Ag nanorods (NRs) (Fig.3D), respectively. After that, these two different morphologies of bimetallic Au@Ag nanocrystals were selfseparated, since the rod-like particles were agglomerated together and precipitated, while the sphere-like particles remained in the supernatant. After separation, the Ag shell was etched and the purified Au NSs (Fig.3E) or Au NBPs (Fig.3F) were obtained. Fig.3G showed the spectra of Au NPs (curve a), where the peaks about 670 and 526 nm were due to the longitudinal plasmon resonance wavelengths (LPRWs) of Au NBPs and the plasmon resonance of Au NSs. Curve b was the spectra for Au@Ag NRs, where the peaks at about 370 and 355 nm were due to the transverse plasmon resonance of Au@Ag NRs and the plasmon reso-

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nance of Ag. The extinction spectrum of purified Au NSs (curve c) and Au NBPs (curve d) overlapped well with the corresponding transverse plasmon peak and longitudinal plasmon peak, respectively. Therefore, Au NBPs with high monodispersity were obtained after purification.

Fig. 3 (A) Schematic purification process of Au NBPs, TEM images of (B) Au NPs, (C) Au@Ag NSs, (D) Au@Ag NRs, (E) Au NSs and (F) Au NBPs, (G) UV-vis spectra of as-prepared Au NPs (a), Au@Ag NRs (b), Au NSs (c) and Au NBPs (d). (H) UV-vis spectra of Au NBPs (a), Au NBPs and AgNO3 (b), mixture of Au NBPs, AAP and AgNO3 without (c) and with (d) ALP, (I, J) TEM images of Au NBPs@Ag with different thicknesses of the silver shell.

The mechanism of the dual-modal PEC and colorimetric immunosensor. Scheme 1A was the schematic illustration of a dual-modal split-type PEC immunosensor with complementary colorimetric detection for MC-LR. Concretely, the antigen was immobilized on the dopaminemodified 96-well microplate. Due to the limited Ab1, the immobilized antigen can compete with free MC-LR in the solution. MSNs were prepared to carry Ab2 for triggering specific immune responses and and S0 for performing HCR. Then, DNA strands (S1 and S2) were added for the in-situ propagation, producing a long DNA complex on the surface of MSNs for signal amplification. After the biotinstreptavidin interaction between biotinylated HCRgenerated DNA and streptavidin-labeled alkaline phosphatase (SA-ALP), ALP can hydrolyze AAP to form AA. In PEC detection, as shown in Scheme 1B, CdS/ZnOHNRs were used as the photoelectric material to modify FTO electrodes. Specifically, CdS as a narrow semiconduc-


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Analytical Chemistry tor with excellent optical absorbability was easily excited to produce photo-induced electron. The electron-hole pairs were generated by the electron jumping from the valence band (VB) to the conduction band (CB) of CdS. Then, photoelectrons rapidly injected into the CB of ZnO HNRs and transferred to FTO for the photocurrent. Notability, the excited holes from ZnO HNRs can jump back to VB of CdS, thus the holes were accumulated and the hole-center was formed. The enzymatic hydrolysate, AA, as an excellent hole-trapping reagent, can effectively prevent the recombination of electrons and holes, thus the photocurrent of CdS/ZnO-HNRs increased and a “signal-on” PEC detection was realized.37-39 In colorimetric detection, the enzymatic hydrolysate, AA, can be also used as the reductant to reduce silver nitrate into silver monomer, which was in-situ deposited on Au NBPs to form Au [email protected] Due to the different thickness of silver shell, the Au NBPs@Ag can show different color change from green to orange with different LSPR peak blue-shift. Thus, a colorimetric detection by naked-eye can be obtained, while quantitative detection can be also achieved by measuring LSPR peak shifts. To demonstrate the feasibility of silver deposition on Au NBPs, a control experiment was carried out. In Fig. 3H (curve a), Au NBPs were green with a longitudinal plasmon peak at 670 nm. When AgNO3 was added into Au NBPs (curve b), the color was slightly shallower but no LPSR peak shift was observed. When Au NBPs were added into the mixture of AgNO3 and AAP (curve c), the color and the LPSR peak didn’t change, too. However, after ALP was introduced (curve d), the color changed from green to orange-red immediately, while the LSPR peak changed from 670 to 556 nm and the peak strength also increased greatly, suggesting the surrounding refractive index of Au NBPs was changed after Ag deposition. The TEM image in Fig.3I implied the walnut-like shape of the bimetallic coreshell Au NBPs@Ag, while a gray color can be found in the inset of Fig. 3I. With more silver deposition, the thickness of the silver shell of Au NBPs@Ag increased gradually, and the color turned to orange-red (Fig.3J). Optimization of the experimental conditions. Some influence factors such as the concentration of AAP, the concentration of AgNO3, the pH of the detection solution and the hydrolysis time of enzyme solution were studied. The concentration of AAP has an important effect on the enzymatic product of AA. In Fig. 4A, the photocurrent intensity increased significantly when the AAP concentration increased. However, when the concentration of AAP was higher than 100 mM, the photocurrent intensity increased slightly, suggesting the optimum concentration of AAP was 100 mM. In addition, the degree of enzymatic hydrolysis depends on the hydrolysis time. Under the optimal concentration of AAP, the photocurrent response increased gradually with the hydrolysis time from 5 to 20 min, owing to the continuous production of AA (Fig. 4B). However, the photocurrent showed no significant change with the prolongation of time. To ensure enzymatic hydrolysis reaction, 20 min was adopted as the optimal hydrolysis time. Furthermore, the pH of the detection solution can also affect the

sensitivity. In Fig. 4C, the photocurrent increased to a maximum at the pH of 8.5 and then decreased, demonstrating that a slightly alkaline environment was suitable. The reason may be that too much acid or too much alkali can cause chemical corrosion of CdS/ZnO-HNRs. Therefore, the optimum pH was set at 8.5. In the colorimetric detection, the concentration of AgNO3 was also important. In Fig. 4D, with the increase in AgNO3 concentration, more silver monomers were reduced and deposited on Au NBPs, resulting in the gradual blue-shift of the LSPR peak from Au NBPs@Ag. However, it reached a platform after the concentration of AgNO3 was higher than 5 mM. Thus, 5 mM AgNO3 was selected.

Fig. 4 Effect of the concentration of AAP (A), hydrolytic time of ALP (B), the pH value of the detection solution (C) and the concentration of AgNO3 (D)

The performance of dual-modal PEC and colorimetric immunosensor. Under optimum conditions, a dual-modal split-type competitive immunosensor with PEC and complementary colorimetric detection was performed for MCLR. For PEC detection (Fig. 5A), the immunosensor without MC-LR can produce higher HCR-generated DNA, then higher ALP to generate AA, and thus the largest photocurrent (Imax). The photocurrent change (∆I) can be obtained by subtracting the photocurrent at various concentrations of MC-LR from Imax. With the increase in MC-LR, less immune complex can be immobilized in the 96-microwell plate, leading to an increase in ∆I. In the range from 0.05 ng/L to 5 µg/L (Fig. 5B), a linear relationship was obtained between ∆I and the logarithm of MC-LR concentrations, while the calibration equation was ∆Ι=121.65+ 19.53×lgCMC-LR (µg/L) with a lower detection limit of 0.03 ng/L (r2=0.991, S/N=3). Simultaneously, a complementary colorimetric detection was also performed by recording the LSPR peak position of Au NBPs@Ag. As shown in Fig. 5C and 5D, with the decrease of MC-LR, the color changed from light green to gray, red, and orange. Meanwhile, the position of LSPR peak varied slowly from 666 to 517 nm and the absorbance increased. In Fig. 5E, the calibration plot also displayed a good linear relationship between the LSPR blue-shifted value (∆λ) and the logarithm of MC-LR concentrations from 0.05 ng/L to 5 µg/L with a lower detection limit of



0.04 ng/L (S/N=3). Besides, the regression equation was ∆λ=127.38+27.41×lgCMC-LR (µg/L) (r2=0.983). Furthermore, compared with other analytical methods of MC-LR

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detection (Table S1), this work exhibited a unique merit of dual-modal signal readout and realized more sensitive and reliable analysis.

Fig. 5. Photocurrent response (A) and UV-vis spectra (D) for different concentrations of MC-LR (a) 5, (b) 1, (c) 0.1, (d) 0.05, (e) 0.01, (f) 0.001, (g) 0.0001, (h) 0.00005, (i) 0 µg/L. Calibration curve based on the photocurrent response (B) and UV-vis spectra (E). (C) Photographs of the immunosensor at different concentrations of MC-LR

Owing to the existence of nonspecific adsorption, the specificity is an essential criterion for immunoassay. So the selectivity of the proposed dual-modal immunosensor was investigated. The possible interferents including MC-LW, MC-LF, MC-RR, MC-YR and Nodularin at the same concentration of 1 µg/L were selected to test. The significant response was only obtained in the presence of MC-LR, and the color was different from other interferents (Fig. 6A). These results indicated the photocurrent changes or the color changes were from the particular association between the immune components, thus the proposed dual-modal immunosensor had a good selectivity. As shown in Fig. 6B, the stability of the CdS/ZnOHNRs modified FTO electrode was studied in electrolyte absence (curve a) and presence (curve b) of AA. After the continuous on/off cycle irradiation for 500 s, the photocurrent kept almost invariable (curve a), indicating the response of CdS/ZnO-HNRs was steady enough for the test. But a slight decrease can be still found due to the photo corrosion of CdS after the long time irradiation. When AA exists in the electrolyte, the photocurrent increased significantly and maintained a more stable tendency (curve b), indicating that

AA can inhibit the photo corrosion of CdS, increase the photocurrent significantly and improve the signal stability. The reproducibility and precision were estimated by inter-assay and intra-assay. Different concentrations of 1, 0.1, 0.01 and 0.001 µg/L MC-LR were applied to study the interassay and RSDs of 2.9%, 3.9%, 3.8% and 5.3% were obtained using one electrode and measuring three times at same condition. The intra-assay was also estimated by analyzing three different electrodes for parallel experiments at 1, 0.1, 0.01 and 0.001 µg/L MC-LR, suggesting the relative standard deviations (RSD) were 7.4%, 7.8%, 6.9% and 7.2%, respectively. Thus, the reproducibility of the immunosensor was acceptable. To estimate the feasibility and application potential in actual analysis, the standard addition method was explored using PEC detection to monitor MC-LR levels in water from tap and lake with different background. The water samples were pretreated merely by filtration to remove large impurities and spiked with 0.001, 0.01, 0.1 and 1 µg/L MC-LR standards. As shown in Fig. 6C, it displayed satisfactory recoveries ranging from 97.9% to 118.9%, implying this proposed novel immunoassay may find great promising potential for practical applications.


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Analytical Chemistry

Fig. 6. (A) Selectivity of the dual-modal immunoassay, (B) Stability evaluation of CdS/ZnO-HNRs/FTO in absence (a) or presence (b) of AA, (C) the recoveries of the detection in water sample.

CONCLUSIONS

In conclusion, an innovative dual-modal split-type immunosensor was constructed for MC-LR detection, basing on the photocurrent change of CdS/ZnO-HNRs and the LSPR blue-shift of Au NBPs@Ag. Such excellent property could be ascribed to the following reasons. (1) The introduction of Ab2-MSNs-S0 conjugate and the amplification of HCR provided the opportunity to load a large number of ALP, which can effectively catalyze AAP. (2) The hydrolysate product, AA, was used as the core element to realize the dual-signal readout by increasing the photocurrent and inducing color change and LSPR peak blue-shift. Compared with traditional PEC immunosensors or colorimetric detections, a more accurate and reliable result can be obtained, benefiting from the different mechanism and relatively independent signal transduction. Therefore, this designed immunoassay not only provides a powerful platform for dual-signal detection of MC-LR, but also has a great potential application in monitoring for other harmful toxins.

ACKNOWLEDGEMENTS

This work was supported by the National Scientific Foundation of China (21475047, U130214, 21705051), the Science and Technology Planning Project of Guangdong Province (2016B030303010), the Scientific Foundation of Guangdong Province (2017A030313077), National Key Research and Development Program of China (SQ2017YFC160089), the Program for the Top Young Innovative Talents of Guangdong Province (2016TQ03N305) and the Foundation for High-level Talents in South China Agricultural University.

ASSOCIATED CONTENT Supporting Information

Materials and instrumentation, Purification of Au NBPs, Adjusting the MSNs, Agarose gel electrophoresis, comparison of available biosensors for analysis of MC-LR. The

Supporting Information is available free of charge on the ACS Publications website.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (Y. Liu). Fax: 86-20-85285026, Phone: 86-20-85280319.

Notes The authors declare no competing financial interest.

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Dual-modal split-type immunosensor for sensitive ... - ACS Publications

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