High-Throughput Signal-On Photoelectrochemical Immunoassay of

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High-Throughput Signal-on Photoelectrochemical Immunoassay of Lysozyme Based on Hole-Trapping Triggered by Disintegrating Bioconjugates of Dopamine-Grafted Silica Nanospheres Xin Li, Xinlei Wang, Lizhi Zhang, and Jingming Gong ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00253 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

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High-Throughput Signal-on Photoelectrochemical Immunoassay of Lysozyme Based on Hole-Trapping Triggered by Disintegrating Bioconjugates of Dopamine-Grafted Silica Nanospheres Xin Li, Xinlei Wang, Lizhi Zhang, and Jingming Gong* †

Key Laboratory of Pesticide & Chemical Biology of Ministry of Education, College

of Chemistry, Central China Normal University, Wuhan 430079, P. R. China

E-mail address: [email protected].

* To whom correspondence should be addressed.

ABSTRACT: A unique split-type photoelectrochemical (PEC) immunoassay has been constructed for detection of low-abundant biocompounds (lysozyme, Lyz, used in this case) via a new trigger strategy by disintegrating bioconjugates of dopamine-grafted silica nanospheres (DA@SiO2NSs) for signal amplification. The preferred electron donor assembly of DA@SiO2NSs is first used as a molecular printboard for positioning anti-Lyz secondary antibody (Ab2) through an amide reaction. With specific immunoreactions in a high-binding microplate, a sandwich immunoassay, the DA@SiO2NSs-based bioconjugate is achieved. By initiating the disintegration of the bioconjugates via acid etching, numerous electron donors of DA are released, thus efficiently triggering hole-trapping with amplified signals obtained. The smart integration of ZnIn2S4-based heterojunctions as photoactive material, a split-type detection mode, and a new trigger strategy by disintegrating the DA@SiO2NSs-based bioconjugate offer an attractive high-throughput signal-on PEC

ACS Paragon Plus Environment

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immunoassay for detection of Lyz. Such an unusual PEC sensor exhibits an outstanding linear response to the concentration in the range between 0.002 and 500 ng mL−1, and the detection limit is as low as 0.6 ppt (S/N = 3). The as-fabricated assay is cost-effective and sensitive. It has been successfully used for measuring Lyz in real samples, which demonstrates the great promise for practical applications.

KEYWORDS: photoelectrochemical immunoassay, high-throughput, bioconjugate

disintegration, dopamine-grafted silica nanosphere, lysozyme

Increasing concerns on health issues, life science, and environmental protection have brought the flourishing development of various analytical assays for ultraselective detection of trace amounts of analytes.1-3 For early clinical diagnosis, disease prevention, and biomedical research, sensitive detection of a disease-related biomarker is becoming increasingly important. A variety of approaches including electrochemistry,4 fluorescence,5 luminescence,6 microfluidic chips,7 naked eye detection,8 surface plasmon resonance9, 10 and photoelectrochemical (PEC) sensors2, 11 have been recently developed for detecting biomarkers. In particular, benefiting from the separation of excitation source and detection signal, PEC immunoassays with remarkable sensitivity, low background signals, cheap instruments, and inherent miniaturization have attracted considerable attention, thus inaugurating an innovative field in biological analysis.12-14 Nowadays, great efforts have been focused on pushing the enhancement of


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detection sensitivity in the field of PEC assay development with more reliability and applicability.15-18 Intrinsically, the PEC signal is closely related with the photon-to-electron conversion of the photoactive materials. During recent decades, various photoactive materials with enhanced photo-to-electron conversion have been explored, enabling the construction of PEC assays.16-18 As a typical ternary chalcogenide semiconductor, ZnIn2S4 (ZIS) is recently becoming attractive in photocatalysis.19,

20

Importantly, ZIS-based heterojunctions with well-defined

nanoarchitectures have shown excellent photocatalytic performance with efficiently refraining the recombination of photo-generated carriers,20-22 which are pregnant and remain a significant challenge to be studied in PEC sensing applications. To meet the increasing demand for ultraselectively detecting biomarkers, another important aspect on further improving PEC assays lies on coupling a signal amplification strategy, i.e. introducing an electron donor, a photogenerated-hole scavenger into the electrolyte. However, the traditional PEC bioassay, subjected to the deficiency of electron donors in living cells or organisms, often directly introduces the electron donor into the electrolyte. This greatly restricts the development of PEC bioassays. To address this issue, a few strategies have been recently proposed by in-situ generating an electron donor. For example, Tang et al. proposed a target-induced nano-enzyme reactor by in-situ generating enzymatic hydrolysate.23 More recently, a PEC immunoassay was constructed using dopamine-loaded liposomes for AFB1 detection.24 Li et al. developed a dopamine-coordinated ligation probe for microRNA assay.25 To further simplify the operation, the elaborate design


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via integrating an electron donor with the PEC sensing system is highly desirable. Owing to the outstanding chemical stability and biocompatibility, silica nanospheres (SiO2NSs), in particular SiO2NSs-based bioconjugates have been widely used for various biomedical applications.26-27 Dopamine (DA), serving as a binder, is bio-adhesive

to

inorganic

and

organic

surfaces.28,

1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride

29

Through (EDC)

a /

N-hydroxysuccinimide (NHS) coupling reaction, catechol groups can be modified on the surface of silica.29,

30

Inspired by this, we construct dopamine-grafted silica

nanospheres (DA@SiO2NSs) as molecular printboards for positioning biomolecules, i.e. used as labeling the detection antibodies. Accompanying the specific antigen-antibody reaction, a sandwich immunoassay can be fabricated with an electron donor (DA) embedded. Once the DA@SiO2NSs-based bioconjugate etched by an aqueous solution of HF, the bioconjugate is disintegrated with DA released. In this case, the released DA is efficient for hole-trapping, triggering the amplification of the photocurrent. Up to now, there has been no report on disintegrating the DA@SiO2NSs-based

bioconjugate

to

amplify

the

photocurrent

for

PEC

immunoassays. In this work we create an unprecedented and split-type PEC immunosensing protocol, whereby ZnO nanoparticles decorated ZnIn2S4 nanoflowers heterojunctions (labeled as ZnONPs-ZISNFs) are coupled with the disintegration of the DA@SiO2NSs-based bioconjugate for triggering the hole-trapping, enabling highly sensitive detection of Lysozyme (Lyz, a ubiquitous protein containing 129 amino


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acids, serving as “body’s own antibiotic”, which is always elevated with the occurrence of related diseases, such as leukemia, meningitis and rheumatoid arthritis31- 34). The split-type detection mode used, i.e. the immunoreaction and signal monitoring separated, effectively eliminates the potential damage of proteins by the illumination, meeting the need of high-throughput analysis. And the heterojunctions of ZnONPs-ZISNFs as photoactive materials facilitate charge separation, thus amplifying the photocurrent response. Meanwhile, the sandwich immunoassay is built using the DA@SiO2NSs-labeled anti-Lyz secondary antibody as the detection antibody (labeled as Ab2-DA@SiO2NSs) in a 96-well microplate. Accompanying the specific antigen-antibody reaction and the etching treatment by HF, the bioconjugate of DA@SiO2NSs-based sandwich immunoassay is disintegrated, resulting in the release of DA. The released DA is able to trap holes efficiently and suppress the electron-hole recombination, thus triggering the amplification of the photocurrent of ZnONPs-ZISNFs (Scheme 1). The as-fabricated system shows a signal-on PEC response upon increasing the concentration of Lyz, via smartly integrating ZnONPs-ZISNFs heterojunctions as the photoactive materials, the split-type detection mode and the fantastic trigger strategy by disintegrating the DA@SiO2NSs-based bioconjugate. To the best our knowledge, this is the first report on the rational combination

of

multiple

functional

components

to

create

an

innovative

high-throughput signal-on PEC immunoassay for the detection of Lyz.

EXPERIMENTAL SECTION Materials and Chemicals. Lysozyme (Lyz), dopamine hydrochloride (DA) and


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1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride

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(EDC)

were

used

without further purification (Aladdin, China). Rabbit monoclonal anti-Lyz antibody (mAb, Jianglai Biological Technology Co., Ltd.) and Bovine serum albumin (BSA, REGAL Biological Technology Co. LTD.) were used as received. Thioacetamide (TAA), succinicanhydride, N-hydroxysuccinimide (NHS), InCl3·4H2O and ZnCl2 were from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). All high-binding polystyrene 96-well microplates were achieved from YEASEN Biological Technology Co., Ltd. (Shanghai, China). Ultrapure water with a resistivity higher than 18 MΩ was used in this work. The buffer solution for measurements is 0.1 M Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl). Apparatus. Scanning electron microscopy (SEM, JSM-5600) was used for studying the general morphology. Fourier Transform Infrared (FTIR) spectra were performed on a Perkin-Elemer PE-983 spectrometer. Ultraviolet-visible (UV-vis) absorption spectra were recorded on a UV-2550 spectrophotometer (Shimadzu). X-ray diffraction (XRD) patterns were collected using an X-ray diffractometer (RigakuUltima III) with high-intensity Cu Kα1irradiation (λ = 1.5406 nm). X-ray photoelectron spectra (XPS) were recorded on a Kratos standard and monochromatic source (Al KR) operated at 15 kV and 10 mA. An electrochemical workstation (CHI 660D, USA) was used to carry out the electrochemical and PEC measurements. The working electrode with the same surface area of 0.125cm2 was used. A Xe lamp of 500 W (Shanghai LanSheng, China) as the radiation source (λ=420 nm) is equipped with a monochromator was used.


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Preparation of ZnONPs-ZISNFs Heterostructures and Photoactive Electrodes. Firstly, nanostructured ZnIn2S4 was synthesized according to the modified procedure35 and then a solution chemical method was used to obtain ZnONPs36 (The experimental details are described in Supporting Information). The composite of ZnIn2S4 nanoflowers decorated with ZnO nanoparticles (ZnONPs-ZISNFs) were synthesized through a post hydrothermal step. In detail, a certain amount of the obtained ZnONPs and ZISNFs with a certain ratio, were mixed and then dispersed in distill water under ultrasonication for 30 min. The resulting mixture was loaded into a stainless steel autoclave (100-mL, Teflon-lined), and heated at 180oC for 4 h. And then the obtained yellow solid products were washed with water and alcohol for several times, dried in vacuum for overnight, and then redispersed into 10 mL of ultrapure water (10 mg mL–1). Finally, a certain amount of the above dispersion of ZnONPs-ZISNFs was dropped onto the clean surface of FTO (labeled as ZnONPs-ZISNFs/FTO). For comparison, the modified electrodes using ZnONPs or ZISNFs separately were also fabricated (designed as ZnONPs/FTO and ZISNFs/FTO). Synthesis of Carboxylic Acid-Functionalized SiO2NSs. Firstly, SiO2NSs as well as surface-functionalized SiO2NSs with amine groups (SiO2NSs-NH2) were prepared by a modified Stöber method (see Supporting Information for details).29,

30, 37

The

obtained SiO2NSs-NH2 were dispersed in a mixture of ethanol and DMF (v : v = 1 : 1), collected by centrifugation, and then redispersed in DMF (50 mL). Following that, the dispersion of SiO2NSs-NH2 was added slowly into 50-mL DMF containing succinic anhydride (0.5 M) under stirring. After it was continuously stirred for 24 h, the


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obtained product via centrifugation was sequentially washed by DMF for twice, a mixture of H2O and DMF in an order of v : v = 1 : 3, 2 : 2 and 3 : 1, and then water, respectively. Finally, surface-functionalized SiO2 NSs with carboxylic acid (SiO2NSs-COOH) were achieved. Synthesis of DA@SiO2NSs-Based Bioconjugates. To prepare the bioconjugate of DA@SiO2NSs-labeled detection antibody, SiO2NSs-COOH was first activated with EDC and NHS to generate NHS ester-terminated SiO2NSs (SiO2NSs-NHS).30,

38

Subsequently, anti-Lyz detection antibody (designated as Ab2, 0.5 mg mL−1, 20 µL) and DA (0.1 M, 40 µL) were added into the above solution (detailed procedures were listed in Supporting Information). The resulting bioconjugate (designed as Ab2-DA@SiO2NSs) was stored in a refrigerator at 4

o

C. The composite of

DA@SiO2NSs was also prepared by the similar procedures without using anti-Lyz detection antibody. Immunoreaction Protocol and Photocurrent Measurements. The immunoreaction occurred in a 96-well polystyrene plate. As capture antibody, the monoclonal anti-Lyz antibody (0.5 mg mL−1 mAb in 0.1 M PBS buffer, pH = 7.0) was first coated in the wells (100 µL per well) at 4oC overnight (designated as Ab1). Then, the plates were rinsed using a buffer solution of 0.05% Tween 20 in 0.1M PBS (pH = 7.0) for three times. In order to block nonspecific binding sites, the plates were further incubated with a blocking solution (0.1M PBS containing 1% BSA, pH = 7.0, 300 µL per well) at 37 oC for 1 h. After the plate was washed as before, Lyz standards or samples (100 µL) at different concentrations in 0.1 M PBS were dropped into the Ab1-modified


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wells and incubated at 37oC for 1 h. Then, the sandwiched immunoassay was achieved by injecting the as-prepared dispersion of Ab2-DA@SiO2NSs bioconjugates (100 µL) into the well and incubating for 1 h. After washing again, 20 µL of 5 wt% HF solution (Vethanol/Vwater = 1) was added into each well,39 thus resulting in the disintegration of the bioconjugates with DA released. Finally, the acidic solution containing the released DA was transferred into a homemade quartz detection cell of 50.0 mL (in 0.1 MTris-HCl, pH = 7.0). A Xe lamp (500 W) as the irradiation source was equipped with a monochromator (λ = 420 nm). The PEC properties were studied by

an

electrochemical

workstation

with

a

three-electrode

system.

The

ZnONPs-ZISNFs/FTO, standard Hg/Hg2Cl2 in saturated KCl solution, and Pt-wire were used as the working, reference, and auxiliary electrode, respectively.

RESULTS AND DISCUSSION Characterization of ZnONPs-ZISNFs Heterostructures. The SEM images for the as-prepared pristine ZISNFs and ZnONPs-ZISNFs are shown in Figure 1. It is observed that ZIS nanoflowers are urchin-shaped and have diameters of 3–5µm (Figure 1A), which are composed of numerous slick nanosheets (Figure 1B). After hydrothermal treatment with ZnONPs, the surface of the nanosheets clearly become rough (Figure 1C). The high-magnification SEM image for the surface of the spheres shows that uniform ZnONPs with a mean size of ~15 nm are distributed onto surface of the ZIS nanosheets (Figure 1D).The XRD patterns for the products are shown in Figure 2A. Curve a can be assigned to a typical hexagonal phase of ZIS (JCPDS 72-0773).40, 41 As shown in curve b, the crystalline ZnONPs could be identified to the


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hexagonal Wurtzite phase (JCPDS 36-1451).42 It can be seen that the composite of ZnONPs-ZISNFs remains the diffraction peaks of pristine ZIS and ZnO, suggesting that the crystal phases of ZIS and ZnO do not change significantly. Also, other peaks for impurities were not observed (curve c). This implies that the composite of ZnONPs/ZIS has been successfully synthesized. Notably, after further hydrothermal treatment with ZnONPs, the crystalline of the as-prepared ZnONPs-ZISNFs is highly improved,

which

is essential to

an

effective

photoactive

material.

The

photoluminescence (PL) spectra and Tyndall effect of the as-prepared ZnONPs dispersion were observed before and after further hydrothermal treatment (Figure S1, Supporting Information). Owing to the colloidal nature of the original ZnONPs dispersion, an intensive emission peak appears at ~540 nm (curve a) and the Tyndall effect (inset of Figure S1) is obvious. After further hydrothermal treatment, the original NPs with small sizes aggregated onto the surface of the ZIS nanosheets, leading to the weaker PL intensity (curve b) and the disappearance of the Tyndall effect. This further suggests that ZnONPs have been attached onto the surface of ZISNFs to form a heterojunction structure through hydrothermal treatment. Characterization of Bioconjugate of Ab2-DA@SiO2NSs. The size and morphology of SiO2NSs before and after decoration were characterized by TEM (Figure S2). It can be seen that the pristine nanostructured SiO2 exhibits a spherical morphology with relatively uniform diameters of ~120 nm. With the formation of the bioconjugate of Ab2-DA@SiO2NSs, the decorated SiO2NSs still maintain the mono-dispersed spherical morphology, but the surface of the SiO2NSs becomes a bit rough. This may


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be due to the decoration of DA and Ab2 onto the surface of SiO2NSs via the amidation reaction. The sequential functionalizations on the surface of silica were evidenced by FT-IR spectra (Figure 2B). The representative absorption peaks for pristine SiO2NS are observed at 1104 cm–1 (asymmetric stretching vibration of Si-O-Si), 955 cm–1 (asymmetric bending and stretching vibration of Si-OH) and 800 cm–1 (stretching vibration of Si-C),30 respectively (curve a). With the amine-modification, the characteristic adsorption peaks of the N-H bending vibration were observed at 1550–1640 cm–1,30 confirming the successful introduction of amine groups onto the SiO2NPs surface (curve b). With further modification using carboxylic groups, a new band appears at 1709 cm–1, originating from the stretching vibration of the C=O bond of carboxylic acid (curve c). After that, followed by the decoration of DA and Ab2 onto the surface of SiO2NSs via the amidation reaction, the C=O bond of carboxylic acid at 1709 cm–1 disappeared, while the C=O bond of amide at 1650 cm–1 was intensified (curve d). In addition, the introduction of DA on SiO2NSs surface was further confirmed by the typical absorption peaks at 1630–1670, 1550–1590 and 1450–1500 cm–1 for the stretching vibration of aromatic C=C,30 especially the typical absorption peaks for the stretching vibration of aromatic C=C introduced by the benzene ring of DA at 1560 cm–1 and 1450 cm–1. The FT-IR results demonstrate that each modification on the surface of silica is successful, finally resulting in the formation of a bioconjugate of Ab2-DA@SiO2NSs via amide reactions. The surface chemical composition of SiO2NSs before and after decoration was


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explored by XPS. Figure 2C shows the survey XPS spectra of pristine SiO2NSs and Ab2-DA@SiO2NSs. It can be seen that several elements such as Si, O, and C exist in pristine SiO2NSs.43, 44 The signal of C is likely resulted from the partially hydrolyzed TEOS.43 After decoration of Ab2-DA onto the surface of SiO2NSs, the presence of N on the surface of Ab2-DA@SiO2NSs was obviously observed. Moreover, the binding energies (EB) values of Si2p, Si2s, C1s and O1s of the bioconjugate are remarkably shifted for 0.05, 0.18, 0.14, and 0.44 eV, respectively (Table S1, Supporting Information), indicating that the local bonding environments are changed, originating from the decoration of Ab2-DA onto the surface of SiO2NSs. The high-resolution XPS spectrum (Figure 2D) indicates the characteristic peak of N 1s at 399.4 and 400.3eV, ascribed to amines -NH2 and amides -N-C=O,44 further confirming the successful decoration of Ab2-DA onto the surface of SiO2NSs. Photoelectrochemical Properties of ZnONPs-ZISNFs/FTO. The photocurrent responses of ZnONPs/FTO, ZISNFs/FTO and ZnONPs-ZISNFs/FTO under visible-light illumination were explored (Figure S3A). Compared with ZnONPs/FTO (curve a) or ZISNFs/FTO (curve b), the PEC response of ZnONPs-ZISNFs/FTO is substantially improved, indicating the evident enhancement of charge separation in the hybrid of ZnONPs-ZISNFs (curve c). It may be assigned to the staggered band potentials of the semiconductors of ZnONPs and ZIS. As shown in Scheme 1, ZISNFs have a negative conduction band (CB) level (~ − 0.73 eV) in comparison with that of ZnONPs (− 0.31 eV). As a result, the photogenerated electrons from ZISNFs are easily injected into the CB of ZnONPs, and move to the external circuit. Meanwhile,


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the holes from the VB of ZnO (~ 2.89 eV) migrate to that of ZIS (~ 1.37 eV). Such a synergistic effect in the designed ZnONPs-ZISNFs could hasten the separation of spatial charges effectively and suppress the recombination of the electron-hole pairs. Thus, the photon-to-electron conversion is increased, which is beneficial to enhance the PEC performance for subsequent immunoassay. With the addition of DA, a hole-trapping reagent, a significantly amplified photocurrent was observed at ZnONPs-ZISNFs/FTO under visible-light irradiation (curve d). As expected, with increasing DA concentration in the solution, the photocurrent response of ZnONPs-ZISNFs/FTO was further increased (curve e), demonstrating the feasibility for the PEC analysis by utilizing the released DA (from the disintegrated bioconjugate) to trigger the photocurrent amplification. The UV-vis diffuse reflectance spectra (DRS) (Figure S3B) also confirm the formation of the ZnONPs-ZISNFs heterojunction. Compared to pristine ZnONPs and ZIS, the plot (αhν)2 versus photo energy (inset of Figure 3B) reveals that the band gap of the ZnONPs-ZISNFs photoelectrode has been narrowed by negative shift of 0.86 and 0.087 eV, respectively, which is attributed to the improved separation efficiency of charge carriers. Optimization of Detection Conditions. As shown in Figure 3A, the weight ratio of ZnONPs to ZISNFs was firstly optimized. It is clear that the photocurrent initially rises with the ZnONPs content increased up to 20% and then decreases. This may be related to a change in the bandgap of the composite of ZnONPs-ZISNFs. With increasing the ZnO content up to 20%, the formed heterojunction between ZISNFs


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and ZnONPs could greatly facilitate the charge separation. While the ZnONPs amount exceeds 20%, the excessive doping of wide-band ZnO in ZnONPs-ZISNFs would result in poor absorption toward visible light. Thus, the optimal weight ratio of ZnONPs was 20%. The amount of photoactive material is also crucial to the PEC responses.

The

photocurrent initially

increases with

the

amount

of the

ZnONPs-ZISNFs dispersion up to 10 µL (10 mg mL−1) and then decreases (Figure 3B). This is due to the nanostructured platform of ZnONPs-ZISNFs on the electrode. With the amount of ZnONPs-ZISNFs further increase, a certain aggregation of nanostructures occurred, hindering the electron transfer with a decreased PEC current. The thicker film may also be easily peeled off from the electrode surface. Thus, the optimal dispersion amount of ZnONPs-ZISNFs is 10 µL. The final PEC response of the decorated electrode was also influenced by the amount of DA (electron donors) released from the bioconjugate of Ab2-DA@SiO2NSs under the aid of HF solution (remove oxygen in advance). With prolonging the reaction time of HF solution in 96 well plates, the photocurrent increased greatly till time up to 20 min and then attenuation (Figure 3C). Initially, with increasing the reaction time, the more DA can be released from the bioconjugate. The released DA severs as an effective hole scavenger, thus leading to the enhanced PEC responses. When further prolonging the reaction time, the released DA is likely to be oxidized by the dissolved oxygen in the solution due to the longer exposure in the air with a result of the decreased photocurrent. Thus, the followed PEC measurements were performed under the optimum reaction time of 20 min.


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The PEC responses of ZnONPs-ZISNFs/FTO are also related to the pH value of the solution. Considering the stability of the photoactive materials of ZnONPs-ZISNFs, the effect of the used pH solution, ranging from 5.0 to 9.0 on the photocurrent of PEC immunoassay was observed using 50 ng mL−1 Lyz as a model. The peak current was maximum at pH 7.0 (Tris-HCl) (Figure3D). Therefore, the pH value of 7.0 was set as an optimal value for the detection solution. Analytical Performance of Split-type Immunoassay for Determination of Lyz. Under the optimized experimental conditions, a bioconjugate disintegration-based immunoassay was applied for PEC detection of Lyz with a split-type detection mode. The photocurrents of ZnONPs-ZISNFs/FTO were determined through the released DA from the disintegrated bioconjugate of Ab2-DA@SiO2NSs, which was related with the concentration of Lyz antigen. As shown in Figure 4A, with the Lyz concentration increase in the solution, the photocurrents increased successively. A linear relationship between the relative change of the photocurrent [(i-i0)/i0, where i0 and I correspond to photocurrent before and after dropping the reaction solution (in the 96 well plate) into the electrolytic cell] and the logarithm of Lyz concentrations was achieved over a range from 0.002 to 500 ng mL−1 (inset of Figure 4A). The corresponding linear equation is: (i-i0)/i0=15.46+5.914log C / ng mL−1 (R = 0.9975). The detection limit is estimated to be 0.0006 ng mL−1 (i.e. 0.6 ppt) at a signal-to-noise ratio of 3σ (σ expressed as the standard deviation of a blank solution, n=6), which was far below the previous reports (shown in Table S2). The outstanding analytical performance could be attributed to the smart integration of the novel


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ZnONPs-ZISNFs heterojunctions as the photoactive materials and a fantastic trigger strategy by disintegrating the bioconjugate of DA@SiO2NSs to amplify the photocurrent signal. In particular, in comparison to the existing methods, the present PEC sensor is cost-effective and promising for in situ and on-site detection of Lyz, because the instruments used in this work are much simpler and common. Moreover, the occurrence of the immunoreactions in 96-well microplate provides a means to simultaneously delivery solution into 96 reservoirs with no cross contamination, precise aliquots of samples, and introduce washing steps for assays. The followed PEC measurements could be finished in nearly 100 s for each well, enabling multiple, simultaneous, replicate assays for high-throughput sensing. Selectivity, Repeatability, and Stability. The selectivity of the as-prepared PEC assay towards Lyz has been investigated. Some possible coexistence in the human serum, such as urea, uric acid, glucose, cysteine, BSA, Alanine and DA, were choosed as the interference. 29 It can be seen that the photocurrent response of each interfering compound alone was close to the background signal (Figure 4B), while the photocurrent response toward the target Lyz was evidently observed. Moreover, in the two-component system containing target Lyz (100 ng mL−1) and individual interfering material (each with 20 fold concentrations, i.e. 2000ng mL−1), the photocurrent response exhibits negligible change. One can see that the photocurrent deviations are all less than 5%, indicating a high specificity of the present PEC immunoassay. In order to observe the repeatability of the present sensor, intra- and inter-assay measurements were carried out. The intra-assay RSD is calculated to be 2.8% (n = 5)


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by using the same-batch sensor for measuring 50 ng mL−1 Lyz. The inter-assay RSD is 4.2 % by using different-batch sensors for five independently measuring 50 ng mL−1 Lyz, suggesting acceptable repeatability. The stability is also crucial to the practical applications. During the initial 10-day storage, the response for Lyz did not obviously decay. After 30 days, the response retained 96% of its initial current value, indicating good stability of the present PEC sensor. Analysis of Human Serum Samples. To further demonstrate the practicality, the present PEC immunoassay was evaluated by measuring real human serum. The serum samples were collected from the normal subject and rheumatoid arthritis patient (from Puren Hospital, Wuhan). Before measurements, the serum sample was treated by centrifugation treatment and diluted 1000-fold. As shown in Table 1, all real samples were spiked at different concentrations of Lyz (0, 50, 100 and 200 ng mL−1, respectively). All recovery values were observed within the range from 92.22% to 104.5 %. The concentration of Lyz in serum Sample 1 (normal subject) was measured to be 3.475 mg L−1, lying in the normal range of 0–6 mg L−1, which agrees well with the previous reports.31, 45 In contrast, the measured value for the concentration of Lyz in serum sample 2 (rheumatoid arthritis patient) was greatly increased to 23.12 mg L−1. In order to demonstrate the accuracy, the PEC results were compared with those obtained by UV-vis spectrophotometer (Figure S4). The serum samples collected from rheumatoid arthritis patient were analyzed using both methods. Compared with a value of 23.12 mg L−1 from the present immunoassay, a value of 19.88 mg L−1 was obtained by UV-vis, showing only a difference of 14.0%, indicating that the present


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method possesses high precision, high accuracy, and good reproducibility. It is feasible for directly analyzing real samples containing the related compounds. Most importantly, the presently proposed PEC assay is cost-effective with an acceptable feasibility to detect Lyz in real human serum samples, without using expensive experimental instruments.

CONCLUSION In summary, a novel signal-on split-type PEC immunoassay has been successfully fabricated for detection of low-abundant protein coupling with ZIS-based heterojunctions as photoactive materials, whereby the preferred electron donor of DA-grafted SiO2NSs is used as molecular printboards, and an fantastic trigger strategy by disintegrating the bioconjugate of Ab2-DA@SiO2NSs is proposed for signal amplification. The disintegration of DA@SiO2NSs-based bioconjugate flexibly triggers hole-trapping with a number of electron donor DA released, thus greatly amplifying the photocurrent response. The developed PEC immunoassay shows high sensitivity and selectivity. The as-fabricated biosensor exhibits fine applicability for sensing Lyz in real serum samples. It is envisioned that this innovative and powerful split-type PEC immunoassay paves a new avenue via introducing a new trigger strategy for signal amplification and offers possibilities in the fields of clinical diagnosis and biomedicines, especially in remote and poor districts short of modern instruments.


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ACKNOWLEDGEMENTS This work was supported by National Science Foundation of China (Grant 21475050, 21777052), and Self-determined Research Funds of CCNU from the Colleges' Basic Research and Operation of MOE (CCNU18TS013).

SUPPORTING INFORMATION AVAILABLE The Supporting Information is available free of charge via the Internet at http://pubs.acs.org. Addition information as noted in the text. Experimental details and Table S1-S2 as well as Figure S1-S4.

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Figure Captions: Scheme 1. Schematic illustration of the principle for PEC detection of Lyz.

Figure 1. SEM images: (A, B) pristine ZISNFs; (C, D) composite ZnONPs-ZISNFs.

Figure 2. (A) XRD patterns of the as-prepared (a) ZISNFs, (b) ZnONPs, and (c) ZnONPs-ZISNFs; (B) FT-IR spectra of (a) pristine SiO2NSs, (b) SiO2NSs-NH2, (c) SiO2NSs-COOH and (d) Ab2-DA@SiO2NSs; (C) XPS survey spectra of (a) SiO2NSs and (b) Ab2-DA@SiO2NSs; (D) High-resolution XPS spectra of N 1s from Ab2-DA@SiO2NSs.

Figure 3. Effects of (A) the loaded content of ZnONPs in the composite of ZnONPs-ZISNFs; (B) volume of ZnONPs-ZISNFs dispersion precasted onto FTO; (C) the reaction time of HF with the bioconjugate of Ab2-DA@SiO2NSs; (D) pH value of Tris-HCl solution on the photocurrent responses (50 ng mL−1 Lyz used in C and D cases).

Figure 4. (A) Photocurrent responses of ZnONPs-ZISNFs/FTO toward Lyz at different concentrations (from bottom to top, 0, 0.002, 0.005, 0.01, 0.02, 0.05, 0.08, 0.1, 0.2, 0.5, 1.0, 5.0, 10, 50, 100 and 500 ng mL−1) in 0.1 M pH 7.0 Tris-HCl. Inset: Linear calibration curve of detection Lyz; (B) Photocurrents response of ZnONPs-ZISNFs/FTO to the interfering substances (2 µg mL−1) exist alone and co-existence with Lyz (100 ng mL−1) with the 0.1M pH 7.0 Tris-HCl as the supporting electrolyte.


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Table 1. PEC Determination of Lyz in Different Serum Samples using the Proposed Method (n=3) Concentration

Recovery (%)

Sample −1

−1

Taken (ng mL )

Found (ng mL )

Negative serum sample 1

0.0

3.475


5.00

53.62

100.3


100.0

95.43

92.22


200.0

197.1

96.87

Positive serum sample1

0.0

23.12

Positive serum sample 2

50.0

67.60

92.45

Positive serum sample 3

100.0

127.6

104.5

Positive serum sample4

200.0

215.5

96.87


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Scheme 1


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Figure 1


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Figure 2


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Figure 3


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Figure 4


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For TOC only


High-Throughput Signal-On Photoelectrochemical Immunoassay of

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