Ultrasensitive Photoelectrochemical Immunoassay for Matrix

Nov 24, 2014 - An ultrasensitive photoelectrochemical sandwich immunoassay was developed to detect matrix metalloproteinase-2 (MMP-2, antigen, Ag) bas...
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Ultrasensitive Photoelectrochemical Immunoassay for Matrix Metalloproteinase‑2 Detection Based on CdS:Mn/CdTe Cosensitized TiO2 Nanotubes and Signal Amplification of SiO2@Ab2 Conjugates Gao-Chao Fan,† Li Han,† Hua Zhu,† Jian-Rong Zhang,*,†,‡ and Jun-Jie Zhu*,† †

State Key Laboratory of Analytical Chemistry for Life Science, School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093, People’s Republic of China ‡ School of Chemistry and Life Science, Nanjing University Jinling College, Nanjing 210089, People’s Republic of China S Supporting Information *

ABSTRACT: An ultrasensitive photoelectrochemical sandwich immunoassay was developed to detect matrix metalloproteinase-2 (MMP-2, antigen, Ag) based on CdS:Mn/ CdTe cosensitized TiO2 nanotubes (TiO2-NTs) and signal amplification of SiO2@Ab2 conjugates. Specifically, the TiO2NTs electrode was first deposited with CdS:Mn by successive ionic layer adsorption and reaction technique and then further coated with CdTe quantum dots (QDs) via the layer-by-layer method, forming TiO2-NTs/CdS:Mn/CdTe cosensitized structure, which was employed as a matrix to immobilize capture MMP-2 antibodies (Ab1); whereas, SiO2 nanoparticles were coated with signal MMP-2 antibodies (Ab2) to form SiO2@Ab2 conjugates, which were used as signal amplification elements via the specific antibody−antigen immunoreaction between Ag and Ab2. The ultrahigh sensitivity of this immunoassay derived from the two major reasons as below. First, the TiO2NTs/CdS:Mn/CdTe cosensitized structure could adequately absorb the light energy, dramatically promote electron transfer, and effectively inhibit the electron−hole recombination, resulting in significantly enhanced photocurrent intensity of the sensing electrode. However, in the presence of target Ag, the immobilized SiO2@Ab2 conjugates could evidently increase the steric hindrance of the sensing electrode and effectively depress the electron transfer, leading to obviously decreased photocurrent intensity. Accordingly, the well-designed photoelectrochemical immunoassay exhibited a low detection limit of 3.6 fg/mL and a wide linear range from 10 fg/mL to 500 pg/mL for target Ag detection. Meanwhile, it also presented good reproducibility, specificity, and stability and might open a new promising platform for the detection of other important biomarkers.

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sensitivity, complicated equipment, and elevated background signals. As a newly emerged and promising analytic method for the detection of biomarkers, photoelectrochemical immunoassay has the features of simple instrument, low cost, and easy miniaturization, which is well-suited for rapid and highthroughput bioanalysis.15 Moreover, it owns potentially higher selectivity because of the totally separated and different energy forms of the excitation source and detection signal resulting in the reduced background signals.16 Hence, photoelectrochemical immunoassay has attracted considerable research interests. Obviously, photoactive materials act as the key factor to analytical performances of the photoelectrochemical biosensors. So far, various semiconductor nanomaterials such as TiO2,17 ZnO,18 CdS,16 CdSe,19 and Bi2S320 have been applied to construct photoelectrochemical biosensors, but most of these

atrix metalloproteinases (MMPs) are a family of zincdependent endopeptidases capable of digesting various extracellular matrix proteins.1 Elevated levels of MMPs have been found to be closely related to pathological development and progression of cancer, which makes the MMPs important biomarkers for early cancer predication and targets for therapeutic drug development.2−4 MMP-2, also called as gelatinase A, is one of the key MMPs due to its ability to degrade type IV collagen.5 Overexpression of MMP-2 has been observed in many different kinds of malignant tumors such as pancreatic cancer,6 colorectal cancer,7 gastric cancer,8 and lung cancer,9 and the expression levels of MMP-2 are often associated with tumor aggressiveness. Accordingly, sensitive detection of MMP-2 expression levels is of great importance to early diagnosis and therapy of the cancers. Previously developed methods for MMP-2 detection include enzymelinked immunosorbent assay (ELISA),10 surface plasmon resonance (SPR),11 fluorescence resonance energy transfer (FRET), 12 bioluminescence resonance energy transfer (BRET),13 and electrochemistry.14 Despite many merits of these methods, some of them involve the drawbacks of limited © 2014 American Chemical Society

Received: October 29, 2014 Accepted: November 24, 2014 Published: November 24, 2014 12398

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Scheme 1. Construction Process of the Photoelectrochemical Sandwich Immunoassay

signal, using SiO2@Ab2 conjugates as signal amplification elements can contribute to an excellent sensitivity for signal-off photoelectrochemical immunoassays. However, this superior signal amplification strategy has not appeared in photoelectrochemical immunoassays until now. Herein, we developed a new platform to construct an ultrasensitive photoelectrochemical immunoassay for MMP-2 (antigen, Ag) detection based on TiO2-NTs/CdS:Mn/CdTe cosensitized structure and signal amplification of SiO2@Ab2 conjugates, as illustrated in Scheme 1. TiO2-NTs electrode obtained via anodic oxidation was first deposited with CdS:Mn by successive adsorption and reaction of Cd2+/Mn2+ and S2− ions and then coated with CdTe film via layer-by-layer assembling oppositely charged polyelectrolyte and CdTe QDs, forming TiO2-NTs/CdS:Mn/CdTe cosensitized structure to significantly enhance the photocurrent intensity. Subsequently, Ab1 was immobilized on the CdTe QDs modified electrode by the classic EDC coupling reaction between amino and carbonyl groups. After BSA blocked unbound sites of the Ab1 modified electrode, the sensing electrode was finished. For target Ag detection, different concentrations of Ag were first bound on the sensing electrode by specific immunoreaction between Ab1 and Ag, and then the fixed concentration of SiO2 @Ab 2 conjugates as signal amplification elements were further bound via specific immunoreaction between Ag and Ab2, leading to significantly decreased photocurrent intensity. The constructed photoelectrochemical immunoassay exhibited ultrahigh sensitivity, reproducibility, specificity, and stability.

sensing systems only involved one or two photoactive species, leading to low photocurrent conversion efficiency. Since the demand for ultrasensitive detection of biomarkers, it is essential to explore effective means to evidently promote the photocurrent response efficiency of semiconductor nanomaterials. To this end, the cosensitized structure, which consists of the large band gap semiconductor coupled with two or more small band gap sensitizers, is highly promising.21 As different semiconductors own different optimal absorption ranges due to their different band gaps, coupling large band gap semiconductor with small band gap sensitizers to produce cosensitized structure with cascade band-edge levels cannot only adequately utilize the light energy but also effectively promote charge separation and consequently enhance the photocurrent conversion efficiency.22 Thus, the cosensitization strategy is an ideal and fascinating choice for constructing photoelectrochemical immunoassays. In terms of signal changes, photoelectrochemical immunoassays can be classified into two types: signal-on and signal-off. As the specific recognition between antibody and antigen would generate a hydrophobic and insulating layer on the sensing electrode which hinders the electron motion, most of the developed photoelectrochemical immunoassays belong to signal-off type. In order to enhance the sensitivity of the immunoassays, enzymes are often utilized as labels linking with secondary antibodies for signal amplification.23−26 However, the introduction of enzyme not only clearly increased the cost and time of sensor preparation but also complicated the testing procedure. As a result, other simple and effective signal amplification strategies will be certainly desirable. To date, SiO2 nanoparticles have received intense interest in biomedical and biological fields owing to their exciting features such as uniform size, large surface area, low toxicity, high biocompatibility, good stability, low cost, etc.27−31 Because of poor electroconductivity and easy modification, SiO2 nanoparticles can be well used as signal-off labels linking with secondary antibodies for signal amplification. Besides, thanks to large surface area, SiO2 nanoparticles can act as carriers for loading multiple secondary antibodies to form secondary antibodies coated SiO2 (SiO2@ Ab2) conjugates. As both the SiO2 nanoparticles and loaded multiple Ab2 jointly facilitate the decrease of photocurrent

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EXPERIMENTAL SECTION

Materials and Reagents. Titanium foil (99.7% purity, 0.127 mm thick), sodium tellurite (Na2TeO3), sodium borohydride (NaBH4), 3-mercaptopropionic acid (MPA), 1ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride (EDC), N-hydroxysuccinimide (NHS), poly(diallyldimethylammonium chloride) (PDDA, 20 wt % in water), and 3-aminopropyl-triethoxysilane (APTS) were all obtained from Sigma-Aldrich. Cadmium nitrate (Cd(NO3)2· 4H2O), manganese acetate (Mn(Ac)2·4H2O), sodium sulfide 12399

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Figure 1. (A) HRTEM image and (B) UV−vis absorption spectrum of the prepared CdTe QDs. Inset of part A: size distribution of the prepared CdTe QDs.

ethanol and treated with 0.4 mL of APTS. After shaking at room temperature for 5 h, the suspension was centrifuged and washed with ethanol three times and DI water twice and then the amino-functionalized SiO2 nanoparticles were obtained. After 0.5 mL of 200 μg/mL Ab2 was activated by 0.5 mL of 10 mg/mL EDC and NHS for 30 min at room temperature, the amino-functionalized SiO2 nanoparticles were dispersed into the above solution. The mixed suspension was incubated for 12 h under shaking at 4 °C and then was blocked by 100 μL of 1% BSA solution for 1 h at room temperature. The unbound Ab2 and BSA were removed by successive centrifugation and washing with DI water several times. Finally, the resulting SiO2@Ab2 conjugates were obtained and dispersed to 2 mL by 10 mM of PBS (pH 7.4). Preparation of TiO2-NTs/CdS:Mn/CdTe Electrode. The TiO2-NTs were prepared by anodic oxidation of a pure titanium sheet according to previous work (see the Supporting Information).33 The deposition of CdS:Mn QDs on TiO2-NTs was according to the successive ionic layer adsorption and reaction (SILAR) technique with some modification.34 The TiO2-NTs electrode was first dipped into 0.1 M Cd(NO3)2 mixed with 0.08 M Mn(Ac)2 methanol solution for 5 min and rinsed with methanol, then followed by dipping into a 0.1 M Na2S methanol/water mixture (1:1, v/v) for 5 min and again rinsed with methanol. This SILAR cycle was repeated six times, and the TiO2-NTs/CdS:Mn electrode was acquired. The CdTe multilayer film was assembled by alternately dipping the TiO2-NTs/CdS:Mn electrode into a 1% PDDA solution and the as-obtained CdTe QDs solution for 10 min, respectively. The film was carefully washed with DI water after each dipping step. The two-step dipping procedure was termed as “one coating”. The coating process was repeated four times, and the TiO2-NTs/CdS:Mn/CdTe electrode was prepared. Immunoassay Development. Ab1 was immobilized onto the TiO2-NTs/CdS:Mn/CdTe electrode via the classic EDC coupling reaction between carbonyl groups on the surface of the MPA-capped CdTe QDs and amino groups of the Ab1. First, the CdTe QDs modified electrode was activated by dropping 25 μL of DI water containing 20 mM EDC and 10 mM NHS for 30 min at room temperature, followed by rinsing with washing buffer solution to remove the excess EDC and NHS. Then 20 μL of 100 μg/mL Ab1 was dropped onto the electrode surface and incubated at 4 °C in a moisture atmosphere overnight. After that, the electrode was rinsed with the washing buffer solution to remove unbound and physically absorbed Ab1. The electrode was then covered with 20 μL of BSA blocking buffer solution for 1 h at room temperature to block nonspecific binding sites and washed with

(Na2S·9H2O), methanol, and hydrofluoric acid (HF, 40 wt % in water) were obtained from Nanjing Chemical Reagent Co., Ltd. (China). SiO2 nanoparticles (99.5%, 30 ± 5 nm) were obtained from Aladdin Reagent Inc. (Shanghai, China). Cadmium chloride (CdCl2·2.5H2O) and sodium hydroxide (NaOH) were purchased from Shanghai Chemical Reagent Co. (China). Ascorbic acid (AA) was purchased from Sinopharm Chemical Reagent Co., Ltd. (China). Matrix metalloproteinase-2 (MMP2, Ag) and matrix metalloproteinase-9 (MMP-9) were obtained from Wuhan Uscn Life Science Inc. (China). Capture MMP-2 antibody (Ab1) and signal MMP-2 antibody (Ab2) were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. (China). Bovine serum albumin (BSA) was purchased from Nanjing Bookman Biotechnology Co. Ltd. (China). Human IgG (HIgG), prostate-specific antigen (PSA), and carcinoembryonic antigen (CEA) were obtained from Shanghai Linc-Bio Science Co. Ltd. (China). All other reagents were of analytical grade and used as received. All aqueous solutions were prepared with deionized water (DI water, 18 MΩ/cm), which was obtained from a Milli-Q water purification system. Phosphate buffer solution (PBS, pH 7.4, 10 mM) was used for the preparation of the antibody and antigen solution, washing buffer solution, and blocking buffer solution which contained 1% (w/v) BSA. Apparatus. Photoelectrochemical measurements were performed with a homemade photoelectrochemical system. A 500 W Xe lamp was used as the irradiation source with the light intensity of 400 μW·cm−2 estimated by a radiometer (Photoelectric Instrument Factory of Beijing Normal University). Photocurrent was measured on a CHI 660D electrochemical workstation (Shanghai Chenhua Apparatus Corporation, China) with a three-electrode system: a modified TiO2-NTs electrode with a geometrical area of 0.25 cm2 as the working electrode, a Pt wire as the counter electrode, and a saturated Ag/AgCl electrode as the reference electrode. The UV−visible (UV−vis) absorption spectra were tested on a UV-3600 UV− visible spectrophotometer (Shimadzu, Japan). Field-emission scanning electron microscopy (FE-SEM) was carried out on a Hitachi S-4800 scanning electron microscope (Hitachi Co., Japan). Transmission electron microscopy (TEM) was performed with a JEOL-2100 transmission electron microscope (JEOL, Japan). Synthesis of CdTe QDs and SiO2@Ab2 Conjugates. Water-soluble MPA-capped CdTe QDs were synthesized based on the literature method with appropriate modifications (see the Supporting Information).32 The typical preparation process for SiO2@Ab2 conjugates was as follows. In total, 10 mg of purchased SiO2 nanoparticles were first dispersed in 2 mL of 12400

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the washing buffer solution thoroughly. Next, 20 μL of different concentrations of target Ag was dropped onto the BSA blocked electrode for an incubation of 1 h at 37 °C followed by washing with washing buffer solution. After the immunoreaction between Ab1 and Ag, the electrode was allowed for labeling by additional incubation with 20 μL of SiO2@Ab2 conjugates solution for 1 h at 37 °C. Finally, the resulting electrode was washed thoroughly with washing buffer solution and introduced into photocurrent measurement. Photoelectrochemical Detection. Photoelectrochemical detection was carried out at room temperature in PBS (pH 7.4, 0.1 M) containing 0.1 M AA, which was served as a sacrificial electron donor during the photocurrent measurement. White light produced by the Xe lamp, with a spectral range of 200− 2500 nm, was utilized as excitation light and was switched on and off every 10 s. The applied potential was 0.0 V. The AA electrolyte was deaerated by pure nitrogen for 15 min before photocurrent measurement.

the typical TEM images of the pure SiO2 nanoparticles (above) and the SiO2@Ab2 conjugates (below). Obviously, the pure SiO2 nanoparticles exhibited uniform size distribution and smooth surfaces with a diameter of about 30 nm, whereas the SiO2@Ab2 conjugates presented rough surfaces with evidently increased external diameter. This difference indicated that multiple Ab2 molecules were successfully decorated on the surface of SiO2 nanoparticles. Photoelectrochemical Property of TiO2-NTs/CdS:Mn/ CdTe Electrode. Highly ordered TiO2-NTs electrode is an excellent substrate material to construct various photoelectrochemical biosensors because of its large surface area, high stability, photoelectric activity, biocompatibility, and low cost.37 However, the wide band gap of TiO2 (∼3.2 eV) limits its direct applications in photoelectrochemical biosensing, since it can only absorb ultraviolet light, leading to the poor utilization of light energy. For highly sensitive detection of biomarkers, the immunosensing system with enhanced photocurrent intensity and less electron−hole recombination is preferred. Modification of TiO2 with different narrow-band gap semiconductors to form cosensitized structure can achieve this goal.38 The band gap of CdS is about 2.4 eV, corresponding to the optimal absorption range of middle-wavelength light, whereas the band gap of CdTe is about 1.7 eV, corresponding to the optimal absorption range of long-wavelength light. Hence, TiO2-NTs coupled with CdS/CdTe can adequately utilize the light energy and significant promote the photocurrent intensity. In order to improve the electron−hole recombination, Mn2+ can be introduced into CdS to form the CdS:Mn doping structure. As the lifetime of electron−hole recombination for CdS:Mn (hundreds of microseconds) is much longer than that of CdS (dozens of nanoseconds),39 the photocurrent intensity would further evidently increase. Accordingly, we hereby first employed TiO2-NTs/CdS:Mn/CdTe cascade cosensitized structure as the photoelectrochemical matrix for the immunosensing electrode. As the doping content of Mn2+, the deposition of CdS:Mn and the thickness of the CdTe film could influence the photocurrent intensity of the TiO2-NTs/ CdS:Mn/CdTe electrode, the optimal preparation conditions were investigated to acquire the maximum photocurrent intensity (see the Supporting Information). Thus, 0.08 M Mn2+, six SILAR cycles of CdS:Mn, and four coating numbers of CdTe QDs were selected to fabricate the TiO2-NTs/ CdS:Mn/CdTe electrode. SEM Characterization of the Immunosensor Fabrication. The surface topography of the TiO2-NTs/CdS:Mn/ CdTe/Ab1/BSA sensing electrode for each fabrication step was characterized by SEM. Figure 3A−D exhibits the typical SEM images of the TiO2-NTs, TiO2-NTs/CdS:Mn, TiO2-NTs/ CdS:Mn/CdTe, and TiO2-NTs/CdS:Mn/CdTe/Ab1/BSA electrode surfaces, respectively. As shown in Figure 3A, highly ordered TiO2 nanotubes with an average inner diameter of about 80 nm and wall thickness of 15 nm have been well fabricated on the titanium sheet. After CdS:Mn deposition, it could be seen that a large quantity of small particles with the size range of 3−7 nm were well distributed on both outside and inside of the nanotube walls, and the wall thickness increased to about 20 nm (Figure 3B). When CdTe QDs were subsequently coated on, as shown in Figure 3C, more small particles adhered to both outside and inside of nanotube walls compared with Figure 3B, and the wall thickness further increased to around 33 nm. After Ab1 and BSA were further immobilized, plenty of larger-sized substances uniformly scattered on the electrode

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RESULTS AND DISCUSSION Characterization of CdTe QDs. Figure 1A,B shows the high-resolution transmission electron microscopy (HRTEM) image and UV−vis absorption spectrum of the synthesized CdTe QDs, respectively. The lattice fringes of the CdTe QDs were clearly shown in the HRTEM image, and the average size of 3.84 nm was obtained from the size distribution in the inset of Figure 1A. The UV−vis absorption spectrum exhibited a broad absorption range below 650 nm and an evident absorption peak at 592 nm. According to Peng’s empirical formula derived from UV−vis absorption,35 the size of the CdTe QDs was calculated to be 3.60 nm. Characterization of SiO2@Ab2 Conjugates. Figure 2 displays the UV−vis spectrum of the formed SiO2@Ab2

Figure 2. UV−vis absorption spectra of (a) Ab 2 , (b) SiO 2 nanoparticles, and (c) SiO2@Ab2 conjugates. The inset is the TEM images of the pure SiO2 nanoparticles (above) and the SiO2@Ab2 conjugates (below).

conjugates. As shown in curve a, the Ab2 solution exhibited a sharp absorption peak located at 280 nm, which corresponded to the characteristic peak of Ab2, generating from π−π* transition in the tryptophan and tyrosine residues of the proteins.36 It could be seen from curve b that the pure SiO2 nanoparticles aqueous suspension did not exhibit any evident absorption peak in the wavelength scope. After SiO2 nanoparticles were coated with Ab2 molecules, an obvious absorption peak located at 280 nm appeared in curve c. Thus, the UV−vis spectra indicated the successful formation of the SiO2@Ab2 conjugates. Besides, the inset of Figure 2 shows 12401

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Figure 3. SEM images of the (A) TiO2-NTs, (B) TiO2-NTs/CdS:Mn, (C) TiO2-NTs/CdS:Mn/CdTe, and (D) TiO2-NTs/CdS:Mn/CdTe/Ab1/ BSA electrode surfaces.

electron−hole recombination. After CdTe QDs were subsequently coated on, the photocurrent intensity (curve c, I = 266.18 μA) increased to 20 times higher than that of the TiO2NTs electrode, which was because the coated CdTe film further increased the absorption range to longer-wavelength light (∼650 nm). Later on, the photocurrent intensity decreased after Ab1 and BSA were successively immobilized on the electrode (curve d, I = 199.61 μA; curve e, I = 168.12 μA), which could be attributed to the steric hindrance of those modified protein molecules. After the as-prepared sensing electrode was incubated with target Ag and then SiO2@Ab2 conjugates, the photocurrent intensity decreased to ∼79% and 37% of the initial sensing electrode (curve f, I = 132.64 μA; curve g, I = 62.55 μA). Notably, the decrement of photocurrent intensity for target Ag modification was evidently less than that of the subsequently modified SiO2@Ab2 conjugates, which was mainly ascribed to obviously increased steric hindrance of the SiO2@Ab2 conjugates. Besides, Ab2 were also used as signal amplification elements to carry out the photoelectrochemical test, and the results exhibited that the decrement of photocurrent response to Ab2 was only about 32% of that to SiO2@Ab2 conjugates, demonstrating the superiority of the SiO2@Ab2 conjugates as signal amplification elements. Thus, the photocurrent responses confirmed the successful formation of the SiO2@Ab2 conjugates and successful construction of the proposed sandwich immunoassay. Of course, the sensitivity of the photoelectrochemical immunoassay depends on the extent of photocurrent change. On the basis of the photocurrent characterization, two major reasons could be concluded to contribute to ultrahigh sensitivity of the proposed sandwich immunoassay, as illustrated in Scheme 2. In the absence of target Ag, the sensing electrode exhibited an evident photocurrent response, because the TiO2-NTs/CdS:Mn/CdTe cascade cosensitized structure could adequately absorb the light energy, dramatically promote the electron transfer, and effectively inhibit the

surface and covered almost all the nanotubes (Figure 3D). Hence, the SEM characterization suggested the successful fabrication of the sensing electrode. Photoelectrochemical Characterization of the Immunoassay. The construction process of the sandwich immunoassay could be monitored by photocurrent responses, as shown in Figure 4. The TiO2-NTs electrode exhibited a small

Figure 4. Photocurrent responses of (a) the TiO2-NTs electrode, (b) after CdS:Mn deposition, (c) after CdTe QDs subsequently coating, (d) after Ab1 immobilization, (e) after BSA blocking, (f) after incubation with 20 μL of 10 μg/mL Ag, and then (g) further incubation with SiO2@Ab2 conjugates.

photocurrent intensity (curve a, I = 12.74 μA), because TiO2 could only harvest ultraviolet light, leading to low photocurrent conversion efficiency. After CdS:Mn QDs were deposited on the TiO2-NTs electrode, the photocurrent intensity (curve b, I = 182.10 μA) increased to 14 times higher than that of the TiO2-NTs electrode, which was because the TiO2-NTs electrode possessed a large surface area for much more CdS:Mn QDs deposition, and the deposited CdS:Mn extended the absorb range to middle-wavelength light (∼570 nm),34 and meanwhile the doped Mn2+ significantly inhibited the 12402

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Scheme 2. Photogenerated Electron−hole Transfer Mechanism of the Sandwich Immunosensor for the Detection of Target Ag

Figure 5. (A) Photocurrent response and (B) calibration curve of the sandwich immunoassay for the detection of different concentrations of target Ag from 10 fg/mL to 500 pg/mL. The error bars showed the standard deviation of five replicate determinations.

Table 1. Analytical Performances of the Recently Developed Photoelectrochemical (PEC) Immunoassaysa

a

immunoassays

analyte

PEC matrix

LOD

ref

label-free PEC detection label-free PEC detection label-free PEC detection label-free PEC detection GOx labeled PEC detection HRP labeled PEC detection CdTe-GOx labeled PEC detection Au@ALP labeled PEC detection Au@HRP labeled PEC detection CdSe labeled PEC detection SiO2@Ab2 labeled PEC detection

mouse IgG pentachlorophenol α-fetoprotein prostate-specific antigen α-synuclein mouse IgG α-fetoprotein prostate-specific antigen prostate-specific antigen human interleukin-6 matrix metalloproteinase-2

CdS TiO2/CdSexTe1−x TiO2/CdS GR-CdS/CoTAPc TiO2/Au CdS TiO2 TiO2/CdS CdS TiO2/CdS TiO2/CdS:Mn/CdTe

8 pg/mL 1 pM 40 pg/mL 0.63 pg/mL 34 pg/mL 0.5 pg/mL 0.13 pg/mL 500 pg/mL 60 fg/mL 0.38 pg/mL 3.6 fg/mL

16 37 42 43 23 24 25 26 44 45 this work

GOx represents glucose oxidase; HRP represents horse radish peroxidase; ALP represents alkaline phosphatase.

monitoring the photocurrent change generated from the amount of the sandwich immunocomplexes, MMP-2 could be detected. Figure 5A presents the photocurrent responses of the immunosensor after being incubated with different concentrations of target Ag and then with fixed concentration of SiO2@Ab2 conjugates. As the concentration of target Ag increased, a greater amount of the large-sized SiO2@Ab2 conjugates were specifically bound on the electrode surface to hinder the electron transfer, leading to gradually decreased photocurrent intensity. As shown in Figure 5B, the photocurrent response linearly decreased with an increasing logarithm of the concentration of target Ag in the range from

electron−hole recombination. However, in the presence of target Ag, the SiO2@Ab2 conjugates specifically bound with target Ag, which evidently increased the steric hindrance of the sensing electrode and effectively depressed the electron transfer from AA electrolyte to the surface of the TiO2-NTs/CdS:Mn/ CdTe cosensitized structure, leading to significantly decreased photocurrent intensity. Photoelectrochemical Detection for MMP-2. MMP-2 detection was based upon the sandwich immunoreactions between Ab1 and target Ag as well as target Ag and SiO2@Ab2 conjugates. The photocurrent response of this immunoassay was directly related to the concentration of target Ag. Thus, by 12403

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10 fg/mL to 500 pg/mL. The regression equation was I = 119.47 − 18.73 log CMMP‑2 (pg/mL), with the correlation coefficient of 0.9971. The limit of detection (LOD, S/N = 3) for target Ag concentration was estimated to be 3.6 fg/mL, which was much lower than those of previous methods such as SPR (36 pg/mL),11 BRET (2 ng/mL),13 gel electrophoresis (1−6 ng/mL),40 upconversion FRET (10 pg/mL),41 electrochemistry (0.11 pg/mL),14 etc. Furthermore, we also listed most of the highly sensitive photoelectrochemical immunoassays toward other biomarkers to illustrate the sensitivity of the proposed immunoassay, as shown in Table 1. It demonstrated that the well-designed immunoassay still exhibited an obvious advantage thanks to the synergy effect of the superior photoelectrochemical property of the TiO2NTs/CdS:Mn/CdTe cosensitized structure and significant signal amplification of the SiO2@Ab2 conjugates. Reproducibility, Specificity, and Stability of the Immunoassay. The reproducibility and precision of the sandwich immunoassay was estimated by both intra-assay and interassay relative standard deviation (RSD). Analyzed from the testing results with five parallel determinations, the intra-assay RSDs were 2.1%, 2.6%, and 3.0% toward 1, 10, and 100 pg/mL of target Ag, respectively. The interassay RSDs of 3.5%, 3.2%, and 2.8% were acquired by detecting the same samples with five electrodes fabricated independently under identical experimental conditions. These results suggested a satisfactory reproducibility and precision of this immunoassay. Specificity is an important criterion for immunoassay, since the nonspecific adsorption can influence the accuracy of the detection results. To confirm that the photocurrent response originated from specific binding of immunoreactions, some representative interfering proteins including matrix metalloproteinase-9 (MMP-9), human IgG (HIgG), prostate-specific antigen (PSA), and carcinoembryonic antigen (CEA) were selected for the interference test. The results indicated that the photocurrent response of the immunoassay was not affected by MMP-9, HIgG, PSA, CEA, and their mixture, as shown in Figure 6. Compared to the photocurrent response tested

demonstrated that the proposed immunoassay had a satisfactory specificity without obvious interference from nonspecific adsorption. The stability of the proposed immunoassay was also investigated. After the sensing electrode was stored in a dark and humid environment at 4 °C over 2 weeks, the photocurrent intensity maintained 95.2% of its initial response, proving the good storage stability.

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CONCLUSIONS In summary, we presented a new promising platform of photoelectrochemical immunoassay for ultrasensitive detection of MMP-2 based on synergy effect of TiO2-NTs/CdS:Mn/ CdTe cosensitized structure and signal amplification of SiO2@ Ab2 conjugates. As the matrix for the sensing electrode, TiO2NTs/CdS:Mn/CdTe cosensitized structure could significantly enhance the photocurrent intensity due to its excellent properties of adequate absorption of light energy, ultrafast electron transfer, and effective inhibition of charge recombination. As the signal amplification elements, SiO 2 @Ab 2 conjugates could significantly reduce the photocurrent intensity because of the obvious steric hindrance effect. Thanks to superior photoelectrochemical property of the TiO2-NTs/ CdS:Mn/CdTe cosensitized structure and prominent signal amplification of the SiO2@Ab2 conjugates, the constructed immunoassay exhibited an ultralow detection limit of 3.6 fg/mL for MMP-2 detection. Since different biomarkers can specifically bind to different antibodies, the proposed photoelectrochemical platform is highly expected to apply in the design of various highly sensitive photoelectrochemical immunoassays, especially for the detection of trace levels of disease-related biomarkers. Moreover, the enhanced cosensitization system also can be extended for probing other important biological interactions such as enzyme biosensing or cell analysis.

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ASSOCIATED CONTENT

S Supporting Information *

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Phone/fax: +86 25 83686130. *E-mail: [email protected]. Phone/fax: +86 25 83597204. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully appreciate the National Natural Science Foundation (Grants 21375059, 21175065, 21335004, and 21121091) and the National Basic Research Program (Grant 2011CB933502) of China.

Figure 6. Photocurrent responses of the immunoassay for 10 pg/mL MMP-2 detection (a) in the absence of interference or in the existence of 100 pg/mL of (b) MMP-9, (c) HIgG, (d) PSA, (e) CEA, and (f) their mixture. The error bars showed the standard deviation of five replicate measurements.

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REFERENCES

(1) Nagase, H.; Woessner, J. F. J. Biol. Chem. 1999, 274, 21491− 21494. (2) Coussens, L. M.; Fingleton, B.; Matrisian, L. M. Science 2002, 295, 2387−2392. (3) Roy, R.; Yang, J.; Moses, M. A. J. Clin. Oncol. 2009, 27, 5287− 5297. (4) Kessenbrock, K.; Plaks, V.; Werb, Z. Cell 2010, 141, 52−67.

without any interfering protein, the relative deviations of the photocurrents response measured in the presence of single interfering protein or their mixture were well within 3.9% corresponding to 10 pg/mL MMP-2 detection. Also the relative standard deviation (RSD) of five parallel determinations for each interference test was within 3.4%. All these results 12404

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dx.doi.org/10.1021/ac504027d | Anal. Chem. 2014, 86, 12398−12405