Biosensors and Bioelectronics 66 (2015) 283–289
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An ultrasensitive and universal photoelectrochemical immunoassay based on enzyme mimetics enhanced signal amplification Guang-Li Wang a,b,n, Jun-Xian Shu a, Yu-Ming Dong a, Xiu-Ming Wu a, Zai-Jun Li a a The Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, PR China b State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing 210093, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 28 August 2014 Received in revised form 18 November 2014 Accepted 18 November 2014 Available online 20 November 2014
An ultrasensitive photoelectrochemical (PEC) immunoassay based on signal amplification by enzyme mimetics was fabricated for the detection of mouse IgG (as a model protein). The PEC immunosensor was constructed by a layer-by-layer assembly of poly (diallyldimethylammonium chloride) (PDDA), CdS quantum dots (QDs), primary antibody (Ab1, polyclonal goat antimouse IgG), and the antigen (Ag, mouse IgG) on an indium-tin oxide (ITO) electrode. Then, the secondary antibody (Ab2, polyclonal goat antimouse IgG) combined to a bio-bar-coded Pt nanoparticle(NP)-G-quadruplex/hemin probe was used for signal amplification. The bio-bar-coded Pt NP-G-quadruplex/hemin probe could catalyze the oxidation of hydroquinone (HQ) using H2O2 as an oxidant, demonstrating its intrinsic enzyme-like activity. High sensitivity for the target Ag was achieved by using the bio-bar-coded probe as signal amplifier due to its high catalytic activity, a competitive nonproductive absorption of hemin and the steric hindrance caused by the polymeric oxidation products of HQ. For most important, the oxidation product of HQ acted as an efficient electron acceptor of the illuminated CdS QDs. The target Ag could be detected from 0.01 pg/mL to 1.0 ng/mL with a low detection limit of 6.0 fg/mL. The as-obtained immunosensor exhibited high sensitivity, good stability and acceptable reproducibility. This method might be attractive for clinical and biomedical applications. & 2014 Elsevier B.V. All rights reserved.
Keywords: CdS quantum dots Bio-bar-coded Pt NP-G-quadruplex/hemin probe Enzyme mimetics Photoelectrochemical immunoassay
1. Introduction The ultrasensitive detection of trace amounts of target protein by immunoassays played an important role in a variety of fields including clinical disease diagnosis (Knox et al., 2011), biomedical research (Ferrari, 2005), food safety (Whitcombe et al., 2011) and environmental protection (Zhu et al., 2011). However, it is still difficult to detect some low-abundance protein biomarkers using conventional methods. Therefore, it is still extremely urgent to develop ultrasensitive methods to satisfy the requirements for the profiling trace amounts of target protein. The photoelectrochemical (PEC) detection is a newly developed but dynamically developing analysis technique for biological assay. It is becoming a new hotspot in analytical chemistry due to its inherent advantages, such as simple equipment, low cost, easy miniaturization and high sensitivity (Wang et al., 2009a). Signal n Corresponding author at: Jiangnan University, School of Chemical and Material Engineering, The Key Laboratory of Food Colloids and Biotechnology, Ministry of Education No.1800, Lihu Road, Wuxi 214122 PR China. E-mail address: [email protected] (G.-L. Wang).
http://dx.doi.org/10.1016/j.bios.2014.11.033 0956-5663/& 2014 Elsevier B.V. All rights reserved.
amplification is the most popular strategy that has been employed to obtain high sensitivity in PEC immunoassays. So far, most of the highly sensitive PEC immunoassays based on the amplified signal strategy were enzyme-labeled immunoassays (Zhao et al., 2012a, 2012b; Sun et al., 2014). Natural enzymes, such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP), were introduced onto a second antibody (Ab2) through immune labeling, which could catalyze the formation of particular products such as the insoluble benzo-4-chlorohexadienone (for HRP) or ascorbic acid (for ALP), inducing an amplified inhibition (for benzo-4-chlorohexadienone) or enhancement (for ascorbic acid) signal in the PEC detection. However, as is known, natural enzymes are sensitive to environmental conditions and can easily be denatured. Furthermore, the preparation, purification and storage of natural enzymes are usually time-consuming and expensive. So, using natural enzymes as labels in PEC immunoassay has limitations. In contrast, artificial enzyme mimetics, the synthesized materials that are able to catalyze the same reaction with that of the natural enzymes, can overcome these shortcomings. Up to now, different enzyme mimetics based on nanomaterials including metal oxide (Mu et al., 2012), carbon (Shi et al., 2011; Wang et al., 2011b) and
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noble metal (Cai et al., 2013) nanostructures were developed. In comparison with natural enzymes, the enzyme mimetics based on nanostructures own the advantages of controlled synthesis in lowcost, high catalytic activity, and high stability against stringent conditions (Lin et al., 2014). For example, Pt nanoparticles (Pt NPs) were demonstrated to have good HRP mimicking activity, which could catalyzed the oxidation of the typical chromogenic substrate such as 3,3′,5,5′-tetramethylbenzidine (TMB) to develop colorimetric sensors (Gao et al., 2013). Hemin is a well-known natural metalloporphyrin (iron protoporphyrin). It is an active cofactor for a variety of enzymes with simpler structure (Li et al., 2007) and it has the peroxidase-like activity similar to that of the natural HRP (Zhang and Dasgupta, 1992). However, direct application of hemin as an oxidative catalyst is a big challenge because of its molecular aggregation in aqueous solution to form catalytically inactive dimers and oxidative self-destruction in the oxidizing media, which cause passivation of its catalytic activity (Bruice, 1991). Luckily, recent research has indicated that hemin conjugated in G-rich DNA sequence (for example G-quadruplex) is a kind of newly discovered HRP mimicking enzyme with higher catalytic activity than that of hemin itself (Kong et al., 2010). The hemin/G-quadruplex DNAzyme can catalyze the oxidation of various substrates including β-nicotinamide adenine dinucleotide hydrogen (NADH) (Zhang et al., 2013a), TMB (Zhang et al., 2011) or luminol (Li et al., 2008a). Due to the enhanced stability and relatively low cost, the hemin/Gquadruplex DNAzyme was recently used as a catalytic label for amplifying biorecognition events with highly efficient signal amplification effect. For example, the hemin/G-quadruplex DNAzyme was implemented as a catalytic label for constructing colorimetric (Du et al., 2011), chemiluminescent (Wang et al., 2011a) and electrochemical (Zhang et al., 2013a; Yuan et al., 2012) biosensors. Despite the successful applications of enzyme mimetics in various analytical methods including electrochemical, colorimetric, chemiluminescent, etc., no report was found for the utilization of enzyme mimetics in PEC sensors. In our experiment, taking mouse IgG (Ag) as a model protein, an ultrasensitive PEC immunosensor based on the signal amplification effect of the enzyme mimetics of bio-bar-coded Pt NP-G-quadruplex/hemin probe was developed. The bio-bar-coded Pt NP-G-quadruplex/ hemin probe as a catalytic amplifier enabled greatly improved sensitivity of the method over the previously reported PEC immunosensors (Zhao et al., 2012b). There are several reasons for the enhanced sensitivity, such as the higher catalytic activity of the bio-bar-coded Pt NP-G-quadruplex/hemin than that of the natural HRP. Especially, different from the report that 4-chloro-1-naphthol (4-CN) (Zhao et al., 2012b) was used as the substrate of natural HRP, hydroquinone (HQ) was explored for the first time as the substrate of the enmyze mimetics in the PEC immunoassay. The oxidation product of HQ was a polymer with abundant electron accepting groups, which could efficiently inhibit the photocurrent of CdS quantum dots (QDs) through photoinduced electron transfer and steric hindrances effect. The photoinduced electron transfer was more exquisite and efficient than the steric hindrances effect (Wang et al., 2009b; Zhao et al., 2012b) commonly used for the construction of PEC immunosenosrs. The detection limit of the PEC immunosensor was lower than most of the previously reported methods (Aziz et al., 2008; Sakharov et al., 2006; Li et al., 2008b; Cowles et al., 2011). This study may open a new avenue for sensitive PEC immunosensing.
2. Experimental 2.1. Chemicals and instrumentation The detailed chemicals and instrumentation are provided in the supplementary information. 2.2. Preparation of Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe The citrate (Cit)-capped Pt NPs were prepared using a previously reported method (Doty et al., 2005) with modifications. The detailed preparation process for Pt NPs is provided in the supplementary information. The bio-bar-coded probe based on Pt NPs and hemin conjugated G-quadruplex DNA was prepared according to a previous report (Lin et al., 2011). The preparation process is shown in Scheme 1A. The detailed preparation of Ab2-bio-bar-coded Pt NPG-quadruplex/hemin probe is provided in the supplementary information. 2.3. Synthesis of water-soluble CdS QDs and the fabrication of modified electrodes Thioglycolic acid (TGA)-stabilized CdS QDs were synthesized using a slightly modified procedure (Wang et al., 2010). The detailed synthetic process for TGA-capped CdS QDs is provided in the supplementary information. The CdS QDs modified ITO electrode was prepared according to a previously reported method (Zhao et al., 2012b). Three layers of CdS QDs were assembled on the ITO electrode and the as obtained electrode was designated as ITO/(PDDA/CdS)3. 2.4. PEC immunosensor construction As shown in Scheme 1B, primary antibody (Ab1, polyclonal goat antimouse IgG) was immobilized onto the CdS QDs modified ITO electrode for the subsequent sandwich-type Ab-Ag (mouse IgG) affinity interactions. The detailed immunoassay construction process is provided in the supplementary information.
3. Results and discussion 3.1. Characterization of CdS QDs and Pt NPs The UV–vis absorption spectrum and the TEM image were used to characterize the as prepared CdS QDs. The UV–vis absorption spectrum showed a clear absorption peak at around 402 nm (Fig. S1A), from which the size of the resulting CdS QDs and its concentration was estimated to be 4.2 nm and 5.5 10 7 mol/L according to Peng’s empirical equations (Yu et al., 2003). The detailed calculation method for the size and concentration of the QDs based on the absorption peak is provided in the supplementary information. The absorption spectrum also implied that the CdS QDs had a broad absorption range that was suitable for the employment as a photoactive substrate in the visible spectrum range. The typical TEM image of the CdS QDs indicated that they were in a near-spherical shape with diameters ranging from 3 to 5 nm (Fig. 1A). The particle size from the TEM image was approximately consistent with that from the result of the above calculation method. The size of the as-synthesized Pt NPs was estimated to be 3–4 nm from the TEM image in Fig. 1B.
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A
B
Scheme 1. Schematic representation of (A) the preparation procedure of Ab2-bio-bar-coded. Pt NP-G-quadruplex/hemin probe, and (B) immunosensor preparation and sandwich-type detection procedure.
3.2. Characterization of Ab2-bio-bar-coded Pt NP-G-quadruplex/ hemin probe The UV–vis absorption spectra of Pt NPs, Ab2-attached Pt NPs, and Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe are shown in Fig. 2. The Pt NPs did not have any characteristic absorption maximum (Gokmen et al., 2009) (Fig. 2, curve a). After the Ab2 molecules were attached onto the surface of Pt NPs, an absorption peak at 270 nm corresponding to the typical protein absorption appeared (curve b), indicating the successful loading of Ab2 on Pt NPs. Compared with the absorption spectrum of Ab2-attached Pt NPs, a new absorption peak at approximately 395 nm appeared for the Ab2-bio-bar-coded Pt NP-G-quadruplex/ hemin probe (curve c). The absorption peak at 395 nm for the probe was close to that of free hemin (394 nm) in solution (Fig. S1B), demonstrating the successful modification of G-quadruplex/ hemin on the Pt NPs. Morever, the absorption peak of Ab2-attached Pt NPs at 270 nm became much sharper and much more intensive after conjugation of the G-quadruplex/hemin
Fig. 2. UV–vis spectra of (a) Pt NPs, (b) Ab2-attached Pt NPs and (c) Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe in 0.01 mol/L PBS (pH 7.4).
Fig. 1. TEM images of (A) CdS QDs and (B) Pt NPs.
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(curve c). This was because the peak of DNA in G-quadruplex also appeared at around 270 nm (Sun, et al., 2013), which coincided with that of the absorption of the protein. 3.3. Characterization of the immunosensor fabrication process It is well-known that electrochemical impedance spectroscopy (EIS) is an effective tool for characterizing the interface properties of electrodes. The value of the diameter of the semicircle at high frequencies reflects the interfacial charge-transfer resistance (Ret). As shown in Fig. S2, the impedance spectrum of bare ITO electrode exhibited a small semicircle due to its good conductivity (curve a, 20 Ω). After the assembly of multilayers of TGA-capped CdS QDs, the electrode surface turned to be negatively charged, which blocked the access of the Fe(CN)63 /4 molecules to the electrode surface, resulting in the increase of the Ret for the electrode (curve b, 115 Ω). After the Ab1 and BSA was immobilized on the electrode surface, the Ret increased further (curve c and d ). The reason for the resistance increase was that the nonconductive properties of the proteins, obstructing electron transfer and mass transport of the electrochemical probe. The EIS results indicated that the antibody was successfully assembled on the electrode surface. Ret further increased to 552 Ω after the specific immunoreactions between antibody and antigen, which was due to steric hindrances of the immunocomplex (curve e). After the subsequent immobilization of Ab2-bio-bar-coded Pt NP-G-Quadruplex/hemin probe, the Ret increased again (curve f, 1104 Ω). The main reason was the steric effect of the immunocomplex and the bio-bar-coded Pt NP-G-Quadruplex/hemin probe, which would further repel the transfer of the negatively charged probe (Fe(CN)63 /4 ). The above results confirmed the successful assembling of the sensing and amplifying elements on the electrode surface. 3.4. Amplified immunoassay based on the Ab2-bio-bar-coded Pt NP– G-quadruplex/hemin probe The fabrication of the immunosensor could also be monitored by PEC experiments. The photocurrent generation mechanism of the CdS QDs modified ITO electrode is shown in Scheme 2. When the CdS QDs absorbed photons with energies higher than that of their band gap, electrons were excited from the valence band (VB) to the conduction
Fig. 3. Photocurrent response of CdS QDs modified electrodes: (a) before and (b) after Ab1 immobilization, (c) after further blocking with BSA, (d) after anchoring the Ag corresponding to 1.0 ng/mL, (e) after further labeling of Ab2-bio-bar-coded Pt NP -G-quadruplex/hemin probe, and (f) after a final 15 min of mimicking enzyme catalyzed oxidation of HQ by H2O2.
band (CB), forming the electron–hole pairs. The electrons transferred to the ITO electrode and generated photocurrent because the energy level of the CB of ITO was lower than that of CdS (Gill et al., 2005). The holes transferred to the surface of the CdS QDs and could be captured by an electron donor (herein was TEA) present in solution. Therefore, the photodissolution reaction and the electron–hole recombination of illuminated CdS QDs were inhibited and the photocurrent intensity could be enhanced (Wang et al., 2009b). Experiment result indicated that a 0.1 mol/L TEA was sufficient to scavenge the photogenerated holes of the CdS QDs (Fig. S3). In the supporting electrolyte of 0.1 mol/ L PBS (pH 7.4) containing 0.1 mol/L TEA, the CdS QDs modified ITO electrode showed an anodic photocurrent under visible light irradiation (2.5 μA, Fig. 3, curve a). After the immobilization of the Ab1 and BSA on the (PDDA/CdS)3 film, the photocurrent intensity decreased (curves b and c). This could be explained by the fact that the immobilization of the insulating proteins on the (PDDA/CdS)3 multilayer modified electrodes sterically hindered the diffusion of the electron donor molecules (TEA) to the surface of CdS for reacting with the photogenerated holes. After the as obtained biosensor was incubated with the corresponding Ag and Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe (curves d and e), the photocurrent further decreased. Similarly, the enhanced steric hindrances resulted from the bio-bar-coded Pt NP-G-quadruplex/hemin and the immunocomplex
Scheme 2. Schematic illustration of the PEC process at CdS QDs-modified ITO electrode in the immunoassay.
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after the biorecognition event hindered the diffusion of TEA to the surface of CdS QDs, leading to photocurrent variation. In addition, hemin itself could meanwhile act as a competitor of light absorption for visible light due to its light-harvesting property at around 320– 430 nm (Fig. S1B) (Han et al., 2013), which weakened the light intensity accessible to excitating the CdS QDs. It was found that not only the Pt NPs but also the G-quadruplex/hemin showed enzyme-like activity, which could catalyze the oxidation of HQ by H2O2 to produce a polymeric product with characteristic absorption peak at around 478 nm (Fig. S4A), whose function was identical to that of the natural enzyme (Zuo et al., 2009). This indicated that both the Pt NPs and the G-quadruplex/ hemin in the Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe contributed to its enzyme-like activity. With the concentrations of the Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe increased, the absorption intensity for the oxidation product of HQ progressively increased and the color of the oxidation product became deeper (Fig. S4B), which implied that the enzyme-like activity of the Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe was catalyst concentration-dependent. The effect of pH on the catalytic activity of the Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe was also studied (Fig. S5). A 0.1 M PBS with pH of 7.4 was used to prepare the solution for the catalytic oxidation of HQ using H2O2 as an oxidant. The enzyme-like activity of the Ab2-bio-bar-coded Pt NP-Gquadruplex/hemin probe was used for signal amplification in PEC detection. It was found that HQ or H2O2 alone or the mixture of HQ and H2O2 caused only a little photocurrent variation of the CdS QDs (Fig. S6). In order to avoid interference from these substrates of the enzyme mimetics, after a final 15 min of mimicking enzyme catalyzed oxidation of HQ by H2O2, the electrodes were rinsed with PBS to remove the excess substrates (such as HQ or H2O2) prior to the photocurrent measurements. Although by doing so, the photocurrent of the detection system decreased clearly after the catalytic reaction between HQ and H2O2 using bio-bar-coded Pt NP-G-quadruplex/hemin as an enzyme mimetics (Fig. 3, curve f). This may point out that the oxidation product of HQ led to the photocurrent decrease of the CdS QDs. As reported, HQ could be oxidized by natural enzymes to form a polymeric product with abundant electron accepting groups (Owsik and Kolarz, 2004; García-Molina et al., 2014). The polymeric product had insulating effect toward the diffusion of TEA to the surface of CdS QDs, diminishing the hole trapping capacity of TEA for CdS QDs and leading to decreased photocurrent. In addition, just like the phenomena we observed previously (Wang et al., 2014), the electron accepting groups of the polymeric products acted as electron acaceptor of the photogenerated electrons of CdS QDs, disrupting the electron transfer from the CB of CdS QDs to the ITO electrode and inhibiting photocurrent generation. In order to confirm the
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photoinduced electron transfer between CdS QDs and the oxidation product of HQ, the fluorescence lifetime of CdS QDs in the absence and presence of the oxidation product of HQ was investigated. As shown in Fig. S7, the fluorescence lifetime of CdS QDs were found to decay in the presence of mimicking enzymatically oxidized HQ. Fitting the fluorescence decay spectra with two decay components gave a major component in 29.6 ns, 94.02% for free CdS QDs, and 0.39 ns, 51.13% in the presence of the oxidation product (Table S1). From these results, it can be concluded that CdS QDs exhibited a significantly shorter fluorescence lifetime in the presence of the oxidation product of HQ, presumably as a result of photo induced electron transfer from CdS QDs to the oxidation products of HQ (Zhang et al., 2013b). 3.5. Optimization of the experimental conditions To achieve an optimal PEC signaling, the incubation time and temperature for the immunoreaction, the amount of the Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe added for the immunoreaction, and the catalytic oxidation time of HQ by the enzyme mimetics were investigated. The results suggested that the maximum response occurred at around 37 °C (Fig. S8A). The lower responses at higher or lower temperatures were attributed to the lower reaction rate at lower temperatures and the instability of binding reagents at higher temperatures. With the increase of incubation time at 37 °C, the photocurrent intensity decreased and then attained a plateau at 60 min (Fig. S8B), indicating that the interaction reached equilibrium. The amount of the Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe added was another important factor for the amplified detection approach. As shown in Fig. S8C, the response rapidly increased with the increased amount of the probe added and then it leveled off when the added probe was above 15 μL. In order to insure a sufficient reaction, 25 μL of the Ab2-bio-bar-coded Pt NP-G-quadruplex/ hemin solution was used in the experiment. Seen from Fig. S8D, the response ascended rapidly with the increase of the catalytic oxidation time up to 15 min, and then tended to be constant. 3.6. Performance of the PEC immunosensor for the target Ag. Under the optimum conditions, the sensitivity of the detection for Ag based on the Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe was investigated. Fig. 4A presented the photocurrents of the CdS QDs modified electrodes after incubation with the Ag of different concentrations. The photocurrent decreased with the increase of the concentration of Ag. The calibration plot showed a good linear relationship between the photocurrent intensity and the logarithm of the analyte concentration in the range from 0.01 pg/mL to 1.0 ng/mL with a correlation coefficient of 0.998. The
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Fig. 4. (A) Effect of different concentrations of Ag on the photocurrent responses. The corresponding calibration curve. Incubation with Ag of elevated concentrations corresponding to (a) 0, (b) 0.01, (c) 0.1, (d) 1, (e) 10, (f)100 and (g) 1000 pg/mL, respectively. (B) Selectivity of the proposed immunoassay to mouse IgG (MIgG) (1.0 ng/mL) by comparing it to the interfering proteins at the 10 ng/mL level: human IgG (HIgG), goat IgG (GIgG), rabbit IgG (RIgG), and the mixed-sample.
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limit of detection (LOD) was 6.0 fg/mL (at a signal-to-noise ratio of 3). This detection limit of this method was much lower than other immunoassays for mouse IgG with different measurement protocols such as electrochemistry (Aziz et al., 2008), chemiluminescent enzyme-linked immunosorbent assay (Sakharov et al., 2006), surface plasmon resonance (Li et al., 2008b), fluorescence (Cowles et al., 2011), surface-enhanced raman spectroscopy (Penn et al., 2013), label-free photoelectrochemistry (Wang et al., 2009b) and HRP-labeled photoelectrochemistry (Zhao et al., 2012b). For a comprehensive literature survey, we lists the analytical performances of various immunoassays for mouse IgG in Table S2. Comparing with the previously reported PEC immunoassay using natural HRP as a catalyst and 4-CN as a substrate (Zhao et al., 2012b), it still exhibited apparent superiority due to the synergy effect existing in this PEC immunoassay. There were several reasons for the improved sensitivity in this amplified PEC immunoassay: (1) The probe exhibited higher catalytic activity than that of natural HRP. As can be seen from Fig. S9, better sensitivity was obtained when using the bio-bar-coded Pt NP-G-quadruplex/ hemin probe than that using HRP. The high catalytic activity resulted from the cooperation effect of Pt NPs and a large number of hemin conjuncated DNA sequences on the bio-bar-coded probe. (2) The most important reason was that the oxidation product of HQ could act as an efficient electron acceptor (Owsik et al., 2004; García-Molina et al., 2014), resulting in the photoinduced electron transfer from the CB of CdS QDs to the LUMO of the oxidation product of HQ (Scheme 2). For the study using 4-CN as a substrate (Zhao et al., 2012b), whose oxidation product was precipates deposited on CdS QDs, no photo induced electron transfer occurred between the oxidation product of 4-CN and CdS QDs. Results confirmed that higher response was obtained in our immunoassay when using HQ as a substrate rather than 4-CN (Fig. S10). (3) The oxidation product of HQ was a polymer (Zuo et al., 2009), which could efficiently block the interaction between the electron donor (TEA) and holes of the excited CdS QDs. We demonstrated the selectivity of the immunoassay using the mouse IgG analogs of goat IgG, rabbit IgG and human IgG.The photocurrent responses of these analogs were much lower than that of mouse IgG (Fig. 4B), demonstrating that the photocurrent reduction was due to the specific binding. The slight photocurrent decrease by the analogs was resulted from nonspecific adsorption. We also measured the responses of mouse IgG in the mixed sample composed of mouse IgG and goat IgG, rabbit IgG, human IgG. Compared to the response in the presence of only mouse IgG, no significant difference in the photocurrent response for the mixed sample was observed. These results demonstrated that this approach exhibited acceptable selectivity for mouse IgG without obvious interference from nonspecific adsorption. The reproducibility of an assay was expressed in terms of values for a within-batch (intraassay) and a between-batch (interassay) relative standard deviation (RSD). The intraassay and interassay RSD obtained from 1.0 ng/mL of mouse IgG were 1.7% and 2.5%, respectively, indicating a good reproducibility of the fabrication protocol. Fig. S11 showed the stability of the photocurrent response of the (PDDA/CdS)3 multilayer film. The irradiation process was repeated for more than 15 times, and the photocurrent was very stable over time without any noticeable decrease. After the QDs modified electrode was stored at 4 °C for two months, the photocurrent intensity did not show obvious change. The results indicated the (PDDA/CdS)3 film in TEA solution possessed high stability and was suitable for the construction of PEC sensors. The feasibility of the immunoassay system for potential clinical applications was investigated using real samples of mouse serum. IgG levels in mouse serum is in the range of 5–13 mg/mL (Penn et al., 2013), which is outside of the liner range of this PEC assay. Therefore, the mouse serum was diluted 1:109 in PBS to reduce IgG
levels to an appropriate concentration. Photocurrent responses to diluted mouse serum sample (diluted 1:109 in PBS) and its subsequent addition of different concentrations of mouse IgG are shown in Fig. S12 in the supplemental information. The recoveries of 1.0, 5.0, 10.0, and 50.0 pg/mL of mouse IgG were determined (shown in Table S3 in the supplemental information) by standard addition methods after deducting IgG photocurrent response from the initially diluted mouse serum. The acceptable recoveries ranging from 91.0% to 109.2% indicated that it was feasible to utilize the developed immunoassay for potentially practical applications.
4. Conclusions A novel strategy using enzyme mimetics of bio-bar-coded nanoparticle probe for signal amplification was developed for the construction of a PEC immunosensor. The bio-bar-coded probe consisted of G-quadruplex-based DNAzyme (hemin conjugated to single-stranded guanine-rich oligonucleotides) attached on the surface of Pt NPs. In comparison with the conventional label-free PEC immunoassays, this immunoassay possessed higher sensitivity because of amplified signal. The amplification effect came from not only the competitive absorption of hemin, but also the higher catalytic activity of the bio-bar-coded probe than that of the natural HRP. Especially, the polymeric oxidation product of HQ blocked directly on the electrode and acted as an electron acceptor of the illuminated CdS QDs. Luckily, both of these two factors inhibited the photocurrent greatly. In a word, this immunoassay showed a wide linear range and acceptable reproducibility, precision, accuracy, indicating the feasible exploitation of enzyme mimetics in versatile PEC immunoreactions or other recognition events.
Acknowledgements This work was supported by The National Natural Science Foundation of China (Nos. 21275065, 21005031), the Fundamental Research Funds for the Central Universities (JUSRP51314B), the MOE & SAFEA for the 111 Project (B13025), and the Opening Foundation of the State Key Laboratory of Analytical Chemistry for Life Science of Nanjing University (KLACLS1008).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.11.033.
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Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios
An ultrasensitive and universal photoelectrochemical immunoassay based on enzyme mimetics enhanced signal amplification Guang-Li Wang a,b,n, Jun-Xian Shu a, Yu-Ming Dong a, Xiu-Ming Wu a, Zai-Jun Li a a The Key Laboratory of Food Colloids and Biotechnology, Ministry of Education, School of Chemical and Material Engineering, Jiangnan University, Wuxi 214122, PR China b State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing 210093, PR China
art ic l e i nf o
a b s t r a c t
Article history: Received 28 August 2014 Received in revised form 18 November 2014 Accepted 18 November 2014 Available online 20 November 2014
An ultrasensitive photoelectrochemical (PEC) immunoassay based on signal amplification by enzyme mimetics was fabricated for the detection of mouse IgG (as a model protein). The PEC immunosensor was constructed by a layer-by-layer assembly of poly (diallyldimethylammonium chloride) (PDDA), CdS quantum dots (QDs), primary antibody (Ab1, polyclonal goat antimouse IgG), and the antigen (Ag, mouse IgG) on an indium-tin oxide (ITO) electrode. Then, the secondary antibody (Ab2, polyclonal goat antimouse IgG) combined to a bio-bar-coded Pt nanoparticle(NP)-G-quadruplex/hemin probe was used for signal amplification. The bio-bar-coded Pt NP-G-quadruplex/hemin probe could catalyze the oxidation of hydroquinone (HQ) using H2O2 as an oxidant, demonstrating its intrinsic enzyme-like activity. High sensitivity for the target Ag was achieved by using the bio-bar-coded probe as signal amplifier due to its high catalytic activity, a competitive nonproductive absorption of hemin and the steric hindrance caused by the polymeric oxidation products of HQ. For most important, the oxidation product of HQ acted as an efficient electron acceptor of the illuminated CdS QDs. The target Ag could be detected from 0.01 pg/mL to 1.0 ng/mL with a low detection limit of 6.0 fg/mL. The as-obtained immunosensor exhibited high sensitivity, good stability and acceptable reproducibility. This method might be attractive for clinical and biomedical applications. & 2014 Elsevier B.V. All rights reserved.
Keywords: CdS quantum dots Bio-bar-coded Pt NP-G-quadruplex/hemin probe Enzyme mimetics Photoelectrochemical immunoassay
1. Introduction The ultrasensitive detection of trace amounts of target protein by immunoassays played an important role in a variety of fields including clinical disease diagnosis (Knox et al., 2011), biomedical research (Ferrari, 2005), food safety (Whitcombe et al., 2011) and environmental protection (Zhu et al., 2011). However, it is still difficult to detect some low-abundance protein biomarkers using conventional methods. Therefore, it is still extremely urgent to develop ultrasensitive methods to satisfy the requirements for the profiling trace amounts of target protein. The photoelectrochemical (PEC) detection is a newly developed but dynamically developing analysis technique for biological assay. It is becoming a new hotspot in analytical chemistry due to its inherent advantages, such as simple equipment, low cost, easy miniaturization and high sensitivity (Wang et al., 2009a). Signal n Corresponding author at: Jiangnan University, School of Chemical and Material Engineering, The Key Laboratory of Food Colloids and Biotechnology, Ministry of Education No.1800, Lihu Road, Wuxi 214122 PR China. E-mail address: [email protected] (G.-L. Wang).
http://dx.doi.org/10.1016/j.bios.2014.11.033 0956-5663/& 2014 Elsevier B.V. All rights reserved.
amplification is the most popular strategy that has been employed to obtain high sensitivity in PEC immunoassays. So far, most of the highly sensitive PEC immunoassays based on the amplified signal strategy were enzyme-labeled immunoassays (Zhao et al., 2012a, 2012b; Sun et al., 2014). Natural enzymes, such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP), were introduced onto a second antibody (Ab2) through immune labeling, which could catalyze the formation of particular products such as the insoluble benzo-4-chlorohexadienone (for HRP) or ascorbic acid (for ALP), inducing an amplified inhibition (for benzo-4-chlorohexadienone) or enhancement (for ascorbic acid) signal in the PEC detection. However, as is known, natural enzymes are sensitive to environmental conditions and can easily be denatured. Furthermore, the preparation, purification and storage of natural enzymes are usually time-consuming and expensive. So, using natural enzymes as labels in PEC immunoassay has limitations. In contrast, artificial enzyme mimetics, the synthesized materials that are able to catalyze the same reaction with that of the natural enzymes, can overcome these shortcomings. Up to now, different enzyme mimetics based on nanomaterials including metal oxide (Mu et al., 2012), carbon (Shi et al., 2011; Wang et al., 2011b) and
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noble metal (Cai et al., 2013) nanostructures were developed. In comparison with natural enzymes, the enzyme mimetics based on nanostructures own the advantages of controlled synthesis in lowcost, high catalytic activity, and high stability against stringent conditions (Lin et al., 2014). For example, Pt nanoparticles (Pt NPs) were demonstrated to have good HRP mimicking activity, which could catalyzed the oxidation of the typical chromogenic substrate such as 3,3′,5,5′-tetramethylbenzidine (TMB) to develop colorimetric sensors (Gao et al., 2013). Hemin is a well-known natural metalloporphyrin (iron protoporphyrin). It is an active cofactor for a variety of enzymes with simpler structure (Li et al., 2007) and it has the peroxidase-like activity similar to that of the natural HRP (Zhang and Dasgupta, 1992). However, direct application of hemin as an oxidative catalyst is a big challenge because of its molecular aggregation in aqueous solution to form catalytically inactive dimers and oxidative self-destruction in the oxidizing media, which cause passivation of its catalytic activity (Bruice, 1991). Luckily, recent research has indicated that hemin conjugated in G-rich DNA sequence (for example G-quadruplex) is a kind of newly discovered HRP mimicking enzyme with higher catalytic activity than that of hemin itself (Kong et al., 2010). The hemin/G-quadruplex DNAzyme can catalyze the oxidation of various substrates including β-nicotinamide adenine dinucleotide hydrogen (NADH) (Zhang et al., 2013a), TMB (Zhang et al., 2011) or luminol (Li et al., 2008a). Due to the enhanced stability and relatively low cost, the hemin/Gquadruplex DNAzyme was recently used as a catalytic label for amplifying biorecognition events with highly efficient signal amplification effect. For example, the hemin/G-quadruplex DNAzyme was implemented as a catalytic label for constructing colorimetric (Du et al., 2011), chemiluminescent (Wang et al., 2011a) and electrochemical (Zhang et al., 2013a; Yuan et al., 2012) biosensors. Despite the successful applications of enzyme mimetics in various analytical methods including electrochemical, colorimetric, chemiluminescent, etc., no report was found for the utilization of enzyme mimetics in PEC sensors. In our experiment, taking mouse IgG (Ag) as a model protein, an ultrasensitive PEC immunosensor based on the signal amplification effect of the enzyme mimetics of bio-bar-coded Pt NP-G-quadruplex/hemin probe was developed. The bio-bar-coded Pt NP-G-quadruplex/ hemin probe as a catalytic amplifier enabled greatly improved sensitivity of the method over the previously reported PEC immunosensors (Zhao et al., 2012b). There are several reasons for the enhanced sensitivity, such as the higher catalytic activity of the bio-bar-coded Pt NP-G-quadruplex/hemin than that of the natural HRP. Especially, different from the report that 4-chloro-1-naphthol (4-CN) (Zhao et al., 2012b) was used as the substrate of natural HRP, hydroquinone (HQ) was explored for the first time as the substrate of the enmyze mimetics in the PEC immunoassay. The oxidation product of HQ was a polymer with abundant electron accepting groups, which could efficiently inhibit the photocurrent of CdS quantum dots (QDs) through photoinduced electron transfer and steric hindrances effect. The photoinduced electron transfer was more exquisite and efficient than the steric hindrances effect (Wang et al., 2009b; Zhao et al., 2012b) commonly used for the construction of PEC immunosenosrs. The detection limit of the PEC immunosensor was lower than most of the previously reported methods (Aziz et al., 2008; Sakharov et al., 2006; Li et al., 2008b; Cowles et al., 2011). This study may open a new avenue for sensitive PEC immunosensing.
2. Experimental 2.1. Chemicals and instrumentation The detailed chemicals and instrumentation are provided in the supplementary information. 2.2. Preparation of Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe The citrate (Cit)-capped Pt NPs were prepared using a previously reported method (Doty et al., 2005) with modifications. The detailed preparation process for Pt NPs is provided in the supplementary information. The bio-bar-coded probe based on Pt NPs and hemin conjugated G-quadruplex DNA was prepared according to a previous report (Lin et al., 2011). The preparation process is shown in Scheme 1A. The detailed preparation of Ab2-bio-bar-coded Pt NPG-quadruplex/hemin probe is provided in the supplementary information. 2.3. Synthesis of water-soluble CdS QDs and the fabrication of modified electrodes Thioglycolic acid (TGA)-stabilized CdS QDs were synthesized using a slightly modified procedure (Wang et al., 2010). The detailed synthetic process for TGA-capped CdS QDs is provided in the supplementary information. The CdS QDs modified ITO electrode was prepared according to a previously reported method (Zhao et al., 2012b). Three layers of CdS QDs were assembled on the ITO electrode and the as obtained electrode was designated as ITO/(PDDA/CdS)3. 2.4. PEC immunosensor construction As shown in Scheme 1B, primary antibody (Ab1, polyclonal goat antimouse IgG) was immobilized onto the CdS QDs modified ITO electrode for the subsequent sandwich-type Ab-Ag (mouse IgG) affinity interactions. The detailed immunoassay construction process is provided in the supplementary information.
3. Results and discussion 3.1. Characterization of CdS QDs and Pt NPs The UV–vis absorption spectrum and the TEM image were used to characterize the as prepared CdS QDs. The UV–vis absorption spectrum showed a clear absorption peak at around 402 nm (Fig. S1A), from which the size of the resulting CdS QDs and its concentration was estimated to be 4.2 nm and 5.5 10 7 mol/L according to Peng’s empirical equations (Yu et al., 2003). The detailed calculation method for the size and concentration of the QDs based on the absorption peak is provided in the supplementary information. The absorption spectrum also implied that the CdS QDs had a broad absorption range that was suitable for the employment as a photoactive substrate in the visible spectrum range. The typical TEM image of the CdS QDs indicated that they were in a near-spherical shape with diameters ranging from 3 to 5 nm (Fig. 1A). The particle size from the TEM image was approximately consistent with that from the result of the above calculation method. The size of the as-synthesized Pt NPs was estimated to be 3–4 nm from the TEM image in Fig. 1B.
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A
B
Scheme 1. Schematic representation of (A) the preparation procedure of Ab2-bio-bar-coded. Pt NP-G-quadruplex/hemin probe, and (B) immunosensor preparation and sandwich-type detection procedure.
3.2. Characterization of Ab2-bio-bar-coded Pt NP-G-quadruplex/ hemin probe The UV–vis absorption spectra of Pt NPs, Ab2-attached Pt NPs, and Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe are shown in Fig. 2. The Pt NPs did not have any characteristic absorption maximum (Gokmen et al., 2009) (Fig. 2, curve a). After the Ab2 molecules were attached onto the surface of Pt NPs, an absorption peak at 270 nm corresponding to the typical protein absorption appeared (curve b), indicating the successful loading of Ab2 on Pt NPs. Compared with the absorption spectrum of Ab2-attached Pt NPs, a new absorption peak at approximately 395 nm appeared for the Ab2-bio-bar-coded Pt NP-G-quadruplex/ hemin probe (curve c). The absorption peak at 395 nm for the probe was close to that of free hemin (394 nm) in solution (Fig. S1B), demonstrating the successful modification of G-quadruplex/ hemin on the Pt NPs. Morever, the absorption peak of Ab2-attached Pt NPs at 270 nm became much sharper and much more intensive after conjugation of the G-quadruplex/hemin
Fig. 2. UV–vis spectra of (a) Pt NPs, (b) Ab2-attached Pt NPs and (c) Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe in 0.01 mol/L PBS (pH 7.4).
Fig. 1. TEM images of (A) CdS QDs and (B) Pt NPs.
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(curve c). This was because the peak of DNA in G-quadruplex also appeared at around 270 nm (Sun, et al., 2013), which coincided with that of the absorption of the protein. 3.3. Characterization of the immunosensor fabrication process It is well-known that electrochemical impedance spectroscopy (EIS) is an effective tool for characterizing the interface properties of electrodes. The value of the diameter of the semicircle at high frequencies reflects the interfacial charge-transfer resistance (Ret). As shown in Fig. S2, the impedance spectrum of bare ITO electrode exhibited a small semicircle due to its good conductivity (curve a, 20 Ω). After the assembly of multilayers of TGA-capped CdS QDs, the electrode surface turned to be negatively charged, which blocked the access of the Fe(CN)63 /4 molecules to the electrode surface, resulting in the increase of the Ret for the electrode (curve b, 115 Ω). After the Ab1 and BSA was immobilized on the electrode surface, the Ret increased further (curve c and d ). The reason for the resistance increase was that the nonconductive properties of the proteins, obstructing electron transfer and mass transport of the electrochemical probe. The EIS results indicated that the antibody was successfully assembled on the electrode surface. Ret further increased to 552 Ω after the specific immunoreactions between antibody and antigen, which was due to steric hindrances of the immunocomplex (curve e). After the subsequent immobilization of Ab2-bio-bar-coded Pt NP-G-Quadruplex/hemin probe, the Ret increased again (curve f, 1104 Ω). The main reason was the steric effect of the immunocomplex and the bio-bar-coded Pt NP-G-Quadruplex/hemin probe, which would further repel the transfer of the negatively charged probe (Fe(CN)63 /4 ). The above results confirmed the successful assembling of the sensing and amplifying elements on the electrode surface. 3.4. Amplified immunoassay based on the Ab2-bio-bar-coded Pt NP– G-quadruplex/hemin probe The fabrication of the immunosensor could also be monitored by PEC experiments. The photocurrent generation mechanism of the CdS QDs modified ITO electrode is shown in Scheme 2. When the CdS QDs absorbed photons with energies higher than that of their band gap, electrons were excited from the valence band (VB) to the conduction
Fig. 3. Photocurrent response of CdS QDs modified electrodes: (a) before and (b) after Ab1 immobilization, (c) after further blocking with BSA, (d) after anchoring the Ag corresponding to 1.0 ng/mL, (e) after further labeling of Ab2-bio-bar-coded Pt NP -G-quadruplex/hemin probe, and (f) after a final 15 min of mimicking enzyme catalyzed oxidation of HQ by H2O2.
band (CB), forming the electron–hole pairs. The electrons transferred to the ITO electrode and generated photocurrent because the energy level of the CB of ITO was lower than that of CdS (Gill et al., 2005). The holes transferred to the surface of the CdS QDs and could be captured by an electron donor (herein was TEA) present in solution. Therefore, the photodissolution reaction and the electron–hole recombination of illuminated CdS QDs were inhibited and the photocurrent intensity could be enhanced (Wang et al., 2009b). Experiment result indicated that a 0.1 mol/L TEA was sufficient to scavenge the photogenerated holes of the CdS QDs (Fig. S3). In the supporting electrolyte of 0.1 mol/ L PBS (pH 7.4) containing 0.1 mol/L TEA, the CdS QDs modified ITO electrode showed an anodic photocurrent under visible light irradiation (2.5 μA, Fig. 3, curve a). After the immobilization of the Ab1 and BSA on the (PDDA/CdS)3 film, the photocurrent intensity decreased (curves b and c). This could be explained by the fact that the immobilization of the insulating proteins on the (PDDA/CdS)3 multilayer modified electrodes sterically hindered the diffusion of the electron donor molecules (TEA) to the surface of CdS for reacting with the photogenerated holes. After the as obtained biosensor was incubated with the corresponding Ag and Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe (curves d and e), the photocurrent further decreased. Similarly, the enhanced steric hindrances resulted from the bio-bar-coded Pt NP-G-quadruplex/hemin and the immunocomplex
Scheme 2. Schematic illustration of the PEC process at CdS QDs-modified ITO electrode in the immunoassay.
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after the biorecognition event hindered the diffusion of TEA to the surface of CdS QDs, leading to photocurrent variation. In addition, hemin itself could meanwhile act as a competitor of light absorption for visible light due to its light-harvesting property at around 320– 430 nm (Fig. S1B) (Han et al., 2013), which weakened the light intensity accessible to excitating the CdS QDs. It was found that not only the Pt NPs but also the G-quadruplex/hemin showed enzyme-like activity, which could catalyze the oxidation of HQ by H2O2 to produce a polymeric product with characteristic absorption peak at around 478 nm (Fig. S4A), whose function was identical to that of the natural enzyme (Zuo et al., 2009). This indicated that both the Pt NPs and the G-quadruplex/ hemin in the Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe contributed to its enzyme-like activity. With the concentrations of the Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe increased, the absorption intensity for the oxidation product of HQ progressively increased and the color of the oxidation product became deeper (Fig. S4B), which implied that the enzyme-like activity of the Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe was catalyst concentration-dependent. The effect of pH on the catalytic activity of the Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe was also studied (Fig. S5). A 0.1 M PBS with pH of 7.4 was used to prepare the solution for the catalytic oxidation of HQ using H2O2 as an oxidant. The enzyme-like activity of the Ab2-bio-bar-coded Pt NP-Gquadruplex/hemin probe was used for signal amplification in PEC detection. It was found that HQ or H2O2 alone or the mixture of HQ and H2O2 caused only a little photocurrent variation of the CdS QDs (Fig. S6). In order to avoid interference from these substrates of the enzyme mimetics, after a final 15 min of mimicking enzyme catalyzed oxidation of HQ by H2O2, the electrodes were rinsed with PBS to remove the excess substrates (such as HQ or H2O2) prior to the photocurrent measurements. Although by doing so, the photocurrent of the detection system decreased clearly after the catalytic reaction between HQ and H2O2 using bio-bar-coded Pt NP-G-quadruplex/hemin as an enzyme mimetics (Fig. 3, curve f). This may point out that the oxidation product of HQ led to the photocurrent decrease of the CdS QDs. As reported, HQ could be oxidized by natural enzymes to form a polymeric product with abundant electron accepting groups (Owsik and Kolarz, 2004; García-Molina et al., 2014). The polymeric product had insulating effect toward the diffusion of TEA to the surface of CdS QDs, diminishing the hole trapping capacity of TEA for CdS QDs and leading to decreased photocurrent. In addition, just like the phenomena we observed previously (Wang et al., 2014), the electron accepting groups of the polymeric products acted as electron acaceptor of the photogenerated electrons of CdS QDs, disrupting the electron transfer from the CB of CdS QDs to the ITO electrode and inhibiting photocurrent generation. In order to confirm the
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photoinduced electron transfer between CdS QDs and the oxidation product of HQ, the fluorescence lifetime of CdS QDs in the absence and presence of the oxidation product of HQ was investigated. As shown in Fig. S7, the fluorescence lifetime of CdS QDs were found to decay in the presence of mimicking enzymatically oxidized HQ. Fitting the fluorescence decay spectra with two decay components gave a major component in 29.6 ns, 94.02% for free CdS QDs, and 0.39 ns, 51.13% in the presence of the oxidation product (Table S1). From these results, it can be concluded that CdS QDs exhibited a significantly shorter fluorescence lifetime in the presence of the oxidation product of HQ, presumably as a result of photo induced electron transfer from CdS QDs to the oxidation products of HQ (Zhang et al., 2013b). 3.5. Optimization of the experimental conditions To achieve an optimal PEC signaling, the incubation time and temperature for the immunoreaction, the amount of the Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe added for the immunoreaction, and the catalytic oxidation time of HQ by the enzyme mimetics were investigated. The results suggested that the maximum response occurred at around 37 °C (Fig. S8A). The lower responses at higher or lower temperatures were attributed to the lower reaction rate at lower temperatures and the instability of binding reagents at higher temperatures. With the increase of incubation time at 37 °C, the photocurrent intensity decreased and then attained a plateau at 60 min (Fig. S8B), indicating that the interaction reached equilibrium. The amount of the Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe added was another important factor for the amplified detection approach. As shown in Fig. S8C, the response rapidly increased with the increased amount of the probe added and then it leveled off when the added probe was above 15 μL. In order to insure a sufficient reaction, 25 μL of the Ab2-bio-bar-coded Pt NP-G-quadruplex/ hemin solution was used in the experiment. Seen from Fig. S8D, the response ascended rapidly with the increase of the catalytic oxidation time up to 15 min, and then tended to be constant. 3.6. Performance of the PEC immunosensor for the target Ag. Under the optimum conditions, the sensitivity of the detection for Ag based on the Ab2-bio-bar-coded Pt NP-G-quadruplex/hemin probe was investigated. Fig. 4A presented the photocurrents of the CdS QDs modified electrodes after incubation with the Ag of different concentrations. The photocurrent decreased with the increase of the concentration of Ag. The calibration plot showed a good linear relationship between the photocurrent intensity and the logarithm of the analyte concentration in the range from 0.01 pg/mL to 1.0 ng/mL with a correlation coefficient of 0.998. The
B
Fig. 4. (A) Effect of different concentrations of Ag on the photocurrent responses. The corresponding calibration curve. Incubation with Ag of elevated concentrations corresponding to (a) 0, (b) 0.01, (c) 0.1, (d) 1, (e) 10, (f)100 and (g) 1000 pg/mL, respectively. (B) Selectivity of the proposed immunoassay to mouse IgG (MIgG) (1.0 ng/mL) by comparing it to the interfering proteins at the 10 ng/mL level: human IgG (HIgG), goat IgG (GIgG), rabbit IgG (RIgG), and the mixed-sample.
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limit of detection (LOD) was 6.0 fg/mL (at a signal-to-noise ratio of 3). This detection limit of this method was much lower than other immunoassays for mouse IgG with different measurement protocols such as electrochemistry (Aziz et al., 2008), chemiluminescent enzyme-linked immunosorbent assay (Sakharov et al., 2006), surface plasmon resonance (Li et al., 2008b), fluorescence (Cowles et al., 2011), surface-enhanced raman spectroscopy (Penn et al., 2013), label-free photoelectrochemistry (Wang et al., 2009b) and HRP-labeled photoelectrochemistry (Zhao et al., 2012b). For a comprehensive literature survey, we lists the analytical performances of various immunoassays for mouse IgG in Table S2. Comparing with the previously reported PEC immunoassay using natural HRP as a catalyst and 4-CN as a substrate (Zhao et al., 2012b), it still exhibited apparent superiority due to the synergy effect existing in this PEC immunoassay. There were several reasons for the improved sensitivity in this amplified PEC immunoassay: (1) The probe exhibited higher catalytic activity than that of natural HRP. As can be seen from Fig. S9, better sensitivity was obtained when using the bio-bar-coded Pt NP-G-quadruplex/ hemin probe than that using HRP. The high catalytic activity resulted from the cooperation effect of Pt NPs and a large number of hemin conjuncated DNA sequences on the bio-bar-coded probe. (2) The most important reason was that the oxidation product of HQ could act as an efficient electron acceptor (Owsik et al., 2004; García-Molina et al., 2014), resulting in the photoinduced electron transfer from the CB of CdS QDs to the LUMO of the oxidation product of HQ (Scheme 2). For the study using 4-CN as a substrate (Zhao et al., 2012b), whose oxidation product was precipates deposited on CdS QDs, no photo induced electron transfer occurred between the oxidation product of 4-CN and CdS QDs. Results confirmed that higher response was obtained in our immunoassay when using HQ as a substrate rather than 4-CN (Fig. S10). (3) The oxidation product of HQ was a polymer (Zuo et al., 2009), which could efficiently block the interaction between the electron donor (TEA) and holes of the excited CdS QDs. We demonstrated the selectivity of the immunoassay using the mouse IgG analogs of goat IgG, rabbit IgG and human IgG.The photocurrent responses of these analogs were much lower than that of mouse IgG (Fig. 4B), demonstrating that the photocurrent reduction was due to the specific binding. The slight photocurrent decrease by the analogs was resulted from nonspecific adsorption. We also measured the responses of mouse IgG in the mixed sample composed of mouse IgG and goat IgG, rabbit IgG, human IgG. Compared to the response in the presence of only mouse IgG, no significant difference in the photocurrent response for the mixed sample was observed. These results demonstrated that this approach exhibited acceptable selectivity for mouse IgG without obvious interference from nonspecific adsorption. The reproducibility of an assay was expressed in terms of values for a within-batch (intraassay) and a between-batch (interassay) relative standard deviation (RSD). The intraassay and interassay RSD obtained from 1.0 ng/mL of mouse IgG were 1.7% and 2.5%, respectively, indicating a good reproducibility of the fabrication protocol. Fig. S11 showed the stability of the photocurrent response of the (PDDA/CdS)3 multilayer film. The irradiation process was repeated for more than 15 times, and the photocurrent was very stable over time without any noticeable decrease. After the QDs modified electrode was stored at 4 °C for two months, the photocurrent intensity did not show obvious change. The results indicated the (PDDA/CdS)3 film in TEA solution possessed high stability and was suitable for the construction of PEC sensors. The feasibility of the immunoassay system for potential clinical applications was investigated using real samples of mouse serum. IgG levels in mouse serum is in the range of 5–13 mg/mL (Penn et al., 2013), which is outside of the liner range of this PEC assay. Therefore, the mouse serum was diluted 1:109 in PBS to reduce IgG
levels to an appropriate concentration. Photocurrent responses to diluted mouse serum sample (diluted 1:109 in PBS) and its subsequent addition of different concentrations of mouse IgG are shown in Fig. S12 in the supplemental information. The recoveries of 1.0, 5.0, 10.0, and 50.0 pg/mL of mouse IgG were determined (shown in Table S3 in the supplemental information) by standard addition methods after deducting IgG photocurrent response from the initially diluted mouse serum. The acceptable recoveries ranging from 91.0% to 109.2% indicated that it was feasible to utilize the developed immunoassay for potentially practical applications.
4. Conclusions A novel strategy using enzyme mimetics of bio-bar-coded nanoparticle probe for signal amplification was developed for the construction of a PEC immunosensor. The bio-bar-coded probe consisted of G-quadruplex-based DNAzyme (hemin conjugated to single-stranded guanine-rich oligonucleotides) attached on the surface of Pt NPs. In comparison with the conventional label-free PEC immunoassays, this immunoassay possessed higher sensitivity because of amplified signal. The amplification effect came from not only the competitive absorption of hemin, but also the higher catalytic activity of the bio-bar-coded probe than that of the natural HRP. Especially, the polymeric oxidation product of HQ blocked directly on the electrode and acted as an electron acceptor of the illuminated CdS QDs. Luckily, both of these two factors inhibited the photocurrent greatly. In a word, this immunoassay showed a wide linear range and acceptable reproducibility, precision, accuracy, indicating the feasible exploitation of enzyme mimetics in versatile PEC immunoreactions or other recognition events.
Acknowledgements This work was supported by The National Natural Science Foundation of China (Nos. 21275065, 21005031), the Fundamental Research Funds for the Central Universities (JUSRP51314B), the MOE & SAFEA for the 111 Project (B13025), and the Opening Foundation of the State Key Laboratory of Analytical Chemistry for Life Science of Nanjing University (KLACLS1008).
Appendix A. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2014.11.033.
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