Redox-Mediated Indirect Fluorescence Immunoassay for the Detection

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Redox-mediated Indirect Fluorescence Immunoassay for the Detection of Disease Biomarkers Using Dopamine-functionalized QDs Wenhui Zhang, Wei Ma, and Yi-Tao Long Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b00048 • Publication Date (Web): 18 Apr 2016 Downloaded from http://pubs.acs.org on April 20, 2016

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Analytical Chemistry

Redox-mediated Indirect Fluorescence Immunoassay for the Detection of Disease Biomarkers Using Dopamine-functionalized QDs Wen-Hui Zhang, Wei Ma*, Yi-Tao Long Key Laboratory for Advanced Materials & Department of Chemistry, East China University of Science and Technology, 130 Meilong Road, Shanghai, 200237, P. R. China ABSTRACT: Here, we report a redox-mediated indirect fluorescence immunoassay (RMFIA) for the detection of the disease biomarker α-fetoprotein (AFP) using dopamine (DA)-functionalized CdSe/ZnS quantum dots (QDs). In this immunoassay, tyrosinase was conjugated with the detection antibody and acted as a bridge connecting the fluorescence signals of the QDs with the concentration of the disease biomarkers. The tyrosinase label used for RMFIA catalyzed the enzymatic oxidation of DAs on the surface of functionalized QDs and caused fluorescence quenching in the presence of the analyte. Using this technique, we obtained a limit of detection as low as 10 pM for AFP. This assay’s potential for clinical analysis was demonstrated by detecting the real sera of patients with hepatocellular carcinoma (HCC). This study makes the first use of RMFIA for the rapid detection of AFP, opening up a new pathway for the detection of disease biomarkers.

According to the most recent data from the Chinese Anti-Cancer Association, cancer is the most common cause of death in China, and nearly 6000 people die of cancer every day in this country. Globally, cancer is set to become one of the main causes of morbidity and mortality in the coming decades in all regions of the world, regardless of the level of resources.1,2 Given this serious situation, the sensitive detection of cancer biomarkers is becoming increasingly significant for early clinical diagnosis, disease prevention and biomedical research.3,4 Significant progress has been made in the detection of disease biomarkers by utilizing, for example, electrochemistry,5,6 fluorescence,7,8 luminescence,9,10 11 12,13 microfluidic chips, naked eye detection and surface plasmon resonance sensors.14 Notably, fluorescence immunoassays have attracted increasing attention because of their high sensitivity, high throughput and efficiency.15-17 The unique optical properties of semiconductor quantum dots (QDs), including their superior photostability, high signal brightness, tunable narrow fluorescence emission and broad absorption spectra, make them attractive candidates in medical areas ranging from bio-imaging and diagnostics to therapy.18,19 Indeed, research on QD-based ﬂuorescence immunoassays (FIAs) for the detection of disease biomarkers has increased dramatically over the past several years.20-22 In these reports, QDs were functionalized with antibodies and acted as fluorescence probes for analytes. However, the complex modification process required to conjugate antibodies with QDs and the various possible antibody conjunct groups on the surface of QDs are often cited as critical barriers to further development of QD-based FIA.23 Thus, obtaining high sensitivity remains challenging. Previous results suggest that QDs are extremely sensitive to the presence of charge on their surfaces or in the surrounding environment. Indeed, this changes the QDs’ optical properties

because the external electrons and holes transfer to either the core conduction band or the surface states of the QDs.24,25 Therefore, modifying the QD surface with a redox substance could be included in the design of fluorescent sensors. For example, Medintz and co-workers reported that QD-dopamine (DA) bioconjugates can function as redox-coupled assemblies for in vitro and intracellular pH sensing.26 Meanwhile, QD-tyrosine has been used for tyrosinase detection.27 The sensing of reactive oxygen species (ROS) and NADH:ubiquinone oxidoreductase has been achieved by QD-ubiquinone bioconjugates via a redox mechanism.28,29 DA is certainly the redox species that is most widely used to modify QDs, and DA-quinone acts as an effective electron acceptor, quenching the fluorescence of QDs. Inspired by these results, we attempted to design an indirect FIA for the detection of disease biomarkers using N-(3, 4-dihydroxyphenethyl)-2-mercaptopentanamide (DAs)-functionalized CdSe/ZnS QDs (DAs-QDs). In this work, instead of QDs, tyrosinase was conjugated with antibodies via a click reaction for immunorecognition. Notably, tyrosinase can act as a bridge connecting the fluorescence signals of DAs-QDs with the concentration of disease biomarkers by catalyzing the oxidation of DAs to DAs-quinone, resulting in fluorescence quenching. The redox-mediated indirect fluorescence immunoassay (RMFIA) was clinically applied and proved to be efficient after detecting 50 real samples. To the best of our knowledge, this is the first report of a fluorescence immunoassay for the rapid detection of α-fetoprotein (AFP) by enzymatic catalysis-induced redox-mediated fluorescence, opening up a new pathway for biomarker detection. In this work, to model the RMFIA in terms of immunoreactions, we employed AFP as our target analyte, with mouse IgM and rabbit IgG as the capture and detection

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antibodies, respectively. To label the enzyme with the detection antibody, tyrosinase and rabbit IgG were conjugated to azide-PEG4-NHS ester (NHS-azide) and dibenzocyclooctyl (DBCO)-PEG4-NHS ester (NHS-DBCO), respectively. These conjugates were then combined by a copper-free click reaction between the DBCO reagent and the azide linker.30 A 96-well microplate was coated with mouse IgM and then sequentially exposed to AFP and tyrosinase-IgG. After washing, DAs-QDs

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were added to the wells. In the presence of AFP, the labeled tyrosinase catalyzes the oxidation of DAs to DAs-quinone and triggers fluorescence quenching; the quenching degree is dependent on the amount of AFP. Thus, the AFP concentration can be determined based on the degree of fluorescence quenching of the QDs during sandwich RMFIA (Figure 1).

Figure 1. Schematic representation of the sandwich RMFIA for the detection of disease biomarkers.

EXPERIMENTAL SECTION Reagents and Materials. All reagents used for all experiments were of analytical grade and were used without further purification. Sodium phosphate monobasic (NaH2PO4, 99%), sodium phosphate dibasic (Na2HPO4, 99%), sodium bicarbonate, sodium carbonate and dimethyl sulfoxide (DMSO, 99.9%) were purchased from Aladdin Chemistry Co., Ltd. Tween-20 was purchased from Acros Organics Co., Ltd. The 96-well polystyrene plates were obtained from Corning Incorporated. Ultrafiltration centrifuge tubes were purchased from Millipore Corporation. DA, bovine serum albumin (BSA), mushroom tyrosinase, and horseradish peroxidase (HRP) were purchased from Sigma-Aldrich Corporation. Rabbit IgG, mouse IgM, and AFP were purchased from Shanghai Yemin Biotech, Inc. NHS-DBCO and NHS-azide were purchased from Click Chemistry Tools Corporation. CdSe/ZnS QDs were purchased from Ocean Nano Tech Corporation. The real samples for AFP clinical diagnosis were provided by the Eastern Hepatobiliary Surgery Hospital. Ultrapure water (18.2 MΩ cm) was obtained from a Millipore system and used for the preparation of all solutions. Apparatus. The fluorescence spectra were acquired with an inverted microscope (eclipse Ti-U, Nikon, Japan) equipped with a super-high-pressure mercury lamp as a light source. Transmission electron microscopy (TEM) images were collected with a JEOL 2100 electron microscope (JEOL Ltd., Japan). Dynamic light scattering (DLS) data were obtained using a Zetasizer Nano ZS (Malvern, United Kingdom). Fourier transform infrared spectroscopy (FTIR) was conducted using a Nicolet-6700 spectrophotometer (Thermo Fisher Scientific, USA). Ultraviolet-visible (UV-Vis) absorption spectra were obtained with an Ocean Optics instrument. Time-of-flight secondary ion mass spectrometry

(TOF-SIMS) m/z spectra were acquired with TOF.SIMS 5 (IONTOF GmbH, Germany). Preparation of DAs-QDs. The DAs-QDs were prepared via the following steps: (1) The surface-capping ligand DAs was designed and synthesized (Supporting Information S1). (2) The DAs ligand was self-assembled onto the QD (λem = 596 nm) surface through the sulfhydryl group. Briefly, QDs were mixed with DAs at different ratios to determine the optimal ratio for the immunoassay (Supporting Information, Figure S8). To prevent the auto-oxidation of DAs on the QD surface, the self-assembly reaction solution was kept at pH 6.5 and degassed with N2 for approximately 10 min before being sealed with sealing film. The reaction mixture was allowed to stand overnight, and then, the prepared DAs-QDs were kept in a refrigerator at 4 °C until use. Preparation and Verification of Tyrosinase-detection Antibody. NHS-DBCO and NHS-azide were first dissolved in dry DMSO before being diluted in their final reaction solutions. Rabbit IgG and tyrosinase were both prepared at a concentration of 10 µM in phosphate-buffered saline (PBS) (pH 7.4). Then, the NHS-DBCO solution (10 mM, 5 µL) was mixed with the IgG solution (10 µM, 100 µL) and the NHS-azide solution (10 mM, 5 µL) was mixed with the tyrosinase solution (10 µM, 100 µL). These two resulting solutions were mixed at a mole ratio of 50:1 and incubated for approximately 30 min at room temperature until the conjugation reactions were complete. Subsequently, quenching buffer (1 M Tris-HCl, pH 8) was added to a final Tris concentration of 50 mM to stop the reactions and promote quenching for 5 min at room temperature. The obtained DBCO-IgG and azide-tyrosinase conjugates were purified with ultrafiltration centrifuge tubes (Amicon Ultra-0.5, Millipore) with a nominal molecular weight limit of 30K.31 Subsequently, the solution of DBCO-IgG (20 µM, 50 µL) was


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added to the solution of azide-tyrosinase (20 µM, 50 µL), and the reaction was kept at room temperature for 2 h. To confirm the success of the conjugation reaction of the obtained tyrosinase-IgG conjugate, 100 µL of AFP diluted 1:300 in coating solution (sodium bicarbonate, 0.2 M, pH 9.4) was added to the wells of 96-well polystyrene plates and incubated overnight at 4 °C. After removing the coating solution and washing the wells three times with PBS containing 0.05% Tween-20 (PBST), blocking buffer (1%

BSA in PBST) was added to the wells to block the remaining protein binding site for 2 h at room temperature. Next, the wells were washed again, and 100 µL of tyrosinase-IgG conjugate was added at different concentrations (100 nM, 60 nM, 10 nM, 1 nM, 100 pM, 10 pM and none) in blocking buffer for 1 h at room temperature. Then, the wells were washed three times, and the prepared DAs-QDs in PBS (pH 6.5, 50 mM, 100 µL) were added to each well and maintained at 37 °C. The fluorescence intensity was recorded after 10 min of incubation.

Figure 2. a) Fluorescence spectra collected from DAs-QDs (red line) and bare QDs (black line) in PBS at a pH of 6.5. b) DLS measurements of the hydrodynamic radii of the QDs (black line) and DAs-QDs (red line). c) FTIR spectra of QDs (black line) and DAs-QDs (red line). d) TEM of DAs-QDs.

Sandwich Assay for AFP. First, 100 µL of mouse IgM diluted 1:300 in coating solution was added into the wells of 96-well polystyrene plates and incubated overnight at 4 °C. The removal and washing steps were executed sequentially, and blocking buffer was added to the wells. After washing again, 100 µL of AFP was added with the desired final concentration from 100 nM to 10 pM in diluent buffer (1% BSA in PBS), and the blank was used as the control (buffer only). After 1 h of incubation, the AFP solutions were removed, and the wells were washed three times with PBST. Then, 100 µL of tyrosinase-IgG conjugate (diluted 1:50 in diluent buffer) was added to the wells and incubated for 1 h at 37 °C. Next, the wells were washed again, and the prepared DAs-QDs was added to each well of the plate and kept at 37 °C for 10 min. The fluorescence intensity was recorded, and error bars were calculated from three independent experiments to represent the standard deviations of the measurements. Real Sample Detection. The serum samples collected from hepatocellular carcinoma (HCC) patients and normal subjects were regarded as positive and negative samples, respectively. The detection procedure for real sera was almost the same as the standard procedure of the sandwich assay for AFP. The only difference was that the AFP was replaced with real serum samples from different patients. Before detection, the amount

of AFP of each serum sample was unknown, and all serum samples were diluted 10 times before being added to the wells. RESULTS AND DISCUSSION Characterization of the DAs-QDs. Upon coating with DAs on the surface of QDs, the fluorescence of the QDs was significantly quenched, indicating the self-assembly of DAs on the QDs’ surfaces (Figure 2a). DLS of the bare QDs in aqueous solution showed that the QDs have an average size of 11.7 nm (Figure 2b, black line). In contrast, the DAs-QDs are slightly larger on average (13.5 nm) (Figure 2b, red line), further demonstrating the successful modification of DAs on QDs. Moreover, the new peak that appeared at 1085.4 cm-1 in the FTIR spectra of the DAs-QDs can be ascribed to the characteristic peak of the C-N stretching vibration of DAs (Figure 2c), which is also consistent with the coating of DAs on the QD surface. Based on the TEM image, the size of the DAs-QDs was approximately 4 nm (Figure 2d), and DLS results, showing that DAs-QDs have a narrow size distribution, also indicated that the functionalized QDs were monodisperse, enabling detection with DAs-QDs. Because DA is easily oxidized, the stabilities of the DAs-QDs and DAs-quinone−QDs were also investigated. As shown in Figure S10, the DAs-QDs resulted in a negligible change in


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the fluorescence intensity (Figure S10a), indicating that DAs remained in the reduced state under the experimental conditions for at least 10 min. Similarly, the QDs with DAs-quinone barely exhibited any fluorescence signal variation (Figure S10b), confirming the stability of this system for the following detection with DAs-QDs. Enzymatic Oxidation Process of Tyrosinase. To maximize the tyrosinase activity, we investigated the conditions of this enzymatic reaction. According to tyrosinase’s product specifications, the optimized pH used in the catalytic oxidation was 6.5. We also investigated the optimal reaction temperature after the addition of 10 µM

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tyrosinase by varying the temperature (Supporting Information, Figure S11). The fluorescence quenching of DAs-QDs is more apparent at 37 °C than at room temperature. Thus, 37 °C was chosen to monitor RMFIA detection. The fluorescence changes of the enzymatic oxidation process at different times (Supporting Information, Figure S12) shows that fluorescence intensity decreases within the first 2 min and plateaus at nearly 10 min. However, there is almost no change in the fluorescence intensity after 10 min, indicating that the maximum generation of DAs-quinone is reached within 10 min. Thus, in this work, the tyrosinase-oxidation process was performed for 10 min in PBS buffer at a pH 6.5 (37 °C).

Figure 3. a) Fluorescence response of DAs-QDs in the presence of various concentrations of tyrosinase in buffer (pH 6.5, 50 mM): (a) blank, (b) 1 pM, (c) 10 pM, (d) 100 pM, (e) 1 nM, (f) 10 nM, (g) 20 nM, (h) 40 nM, (i) 60 nM, (j) 80 nM, (k) 100 nM, (l) 1 µM, and (m) 10 µM. b) The fluorescence intensity exhibited an approximately linear decrease as the concentration of tyrosinase increased.

Availability of RMFIA System. As shown in Figure 3a, the fluorescence intensity of the DAs-QDs gradually increased as the concentration of tyrosinase decreased from 10 µM to 1 pM; below a certain concentration, the fluorescence was not quenched further (Supporting Information, Figure S13). This indicates that DAs-quinone is generated on the surface of the QD conjugates via the enzymatic-oxidation of DAs by the catalysis of tyrosinase. Following the photoexcitation of DAs-QDs, the QD’s conduction band electron is transferred to the lowest unoccupied molecular orbital of the DAs-quinone acceptor and then shuttled back to the QD’s valence band through nonradiative pathways (Figure 1). Thus, DAs-quinone exhibits surface-related trap states that act as fast nonradiative de-excitation routes for photoinduced electron carriers, resulting in fluorescence quenching. The quenching degree is related to the tyrosinase concentration. As depicted in Figure 3b, the relative fluorescence intensity (I/I0) of the DAs-QDs showed good linearity relative to the logarithm of the tyrosinase concentration from 10 µM to 10 pM, and the DAs-QDs’ detection limit for tyrosinase was as low as 10 pM. To further investigate the biospecificity of the RMFIA system, BSA and HRP were used as controls. No obvious changes were noted when HRP and BSA were added to the DAs-QDs, confirming that the fluorescence quenching results from the tyrosinase-catalyzed oxidation of DAs to DAs-quinone (Supporting Information, Figure S14). Verification of the Tyrosinase-detection Antibody Conjugate. To demonstrate the successful conjugation of tyrosinase and rabbit IgG, antigen diluted in coating solution was added to the wells of 96-well polystyrene plates and incubated overnight at 4 °C. After washing and blocking steps, 100 µL of different concentrations of tyrosinase-IgG conjugate in blocking buffer was added. Then, the plates were washed three times, and the prepared DAs-QDs were added to each well and maintained at 37 °C for 10 min. The resulting

fluorescence intensity decreased as the tyrosinase-IgG concentration increased from 10 pM to 100 nM (Figure 4). However, the fluorescence intensity of each well showed almost no change when different concentrations of unconjugated tyrosinase were incubated (Supporting Information, Figure S15). Moreover, new peaks (204: C10H20O4+, 219: C10H21NO4+) were detected by TOF-SIMS when tyrosinase was conjugated with rabbit IgG (Supporting Information, Figure S16, S17). All of these results confirmed that tyrosinase and IgG were successfully conjugated.

Figure 4. Fluorescence response of DAs-QDs treated with different amounts of tyrosinase-IgG: (a) blank, (b) 10 pM, (c) 100 pM, (d) 1 nM, (e) 10 nM, (f) 60 nM, and (g) 100 nM.

Fluorescence Detection of AFP with the RMFIA. After demonstrating the successful conjugation of tyrosinase with rabbit IgG, we utilized this conjugate as the bridge to connect the fluorescence signals of the DAs-QDs with the amount of target analyte in the RMFIA. Here, we used AFP as our model


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disease biomarker. The 96-well plate was modified with mouse IgM and then blocked with 1% BSA-PBS solution. Serial dilutions of AFP (100 nM to 10 pM) were added to the wells, followed by the tyrosinase-IgG conjugates. Finally, the DAs-QDs were added to the wells, which were incubated at 37 °C for 10 min. Figure 5 shows that the fluorescence intensity at 596 nm gradually decreased as the AFP concentration increased. The fluorescence change was attributed to the biocatalytic reaction of DAs, which enabled the production of DAs-quinone for quenching the fluorescence because of the electron transfer from QD to DAs-quinone. The inset in Figure 5 shows that the relative fluorescence intensity (I/I0) of the DAs-QDs exhibited good linearity with AFP from 100 nM to 10 pM. The DAs-QDs’ detection limit for AFP was 10 pM, whereas the AFP concentration in normal subjects is less than 200 pM. Thus, this method could be useful for the sensitive detection of disease biomarkers in clinical applications.

Figure 5. Fluorescence response of DAs-QDs to various concentrations of AFP: (a) blank, (b) 10 pM, (c) 100 pM, (d) 1 nM, (e) 10 nM, (f) 60 nM, and (g) 100 nM. The inset shows that the fluorescence intensity exhibited an approximately linear decrease as the AFP concentration increased.

with the RMFIA to determine the possible interferences affecting detection in real samples. For this purpose, 40 different AFP-positive samples collected from clinical patients with HCC were analyzed, and 10 other AFP-negative samples were used as controls. The experiment was performed on the 50 unrelated samples after a 10-fold dilution. Compared with the AFP concentration determined in the hospital (Supporting Information, Table S1), higher AFP concentrations (positive samples) generated low values of I/I0, whereas lower AFP concentrations (negative samples) yielded high values of I/I0. The RMFIA was able to differentiate positive and negative samples based on I/I0 values, and the threshold is shows by the red line in Figure 6a. Out of a total of 40 samples from patients with diagnosed HCC, only one sample gave as a false negative, and all of the 10 control samples were negative (Figure 6a). As shown in Figure 6b, the receiver operating characteristic (ROC) curve demonstrated that our RMFIA had a sensitivity of 97.5% and a specificity of 100% in one independent experiment. The area under the ROC curve was calculated to be approximately 0.98, confirming the high accuracy of this RMFIA. Thus, these results verify that our assay could be used to determine AFP in real samples. CONCLUSIONS We have developed a RMFIA for AFP detection by tyrosinase-induced fluorescence quenching using DAs-QDs. This is the first report of the detection of disease biomarkers by redox-mediated fluorescence changes. In this work, tyrosinase was conjugated with a detection antibody and acted as a bridge connecting the QDs’ fluorescence signals and the concentration of disease biomarkers. The tyrosinase label used for the RMFIA catalyzes the enzymatic oxidation of DAs on the surface of functionalized QDs, resulting in decreased fluorescence intensity in the presence of the analyte. AFP concentrations as low as 10 pM could be detected based on fluorescence changes using our RMFIA. We believe that the proposed method has good potential for extension to other applications after further development. Overall, our approach provides a new pathway for achieving versatile and sensitive detection by RMFIA. Future efforts will be directed toward increasing the RMFIA’s sensitivity and selectivity.

Real Sample Detection of AFP. Encouraged by the sensitivity of this RMFIA, we performed clinical diagnoses

Figure 6. AFP detection results in human sera with the RMFIA. The assay was applied to detect AFP in sera from clinical patients. Clinical samples were collected from patients with HCC and 10 normal subjects (control) were used to evaluate the detection performance of the RMFIA. a) The signal-to-cutoff ratios for positive and negative samples. b) ROC curve describing the sensitivity and specificity of the RMFIA for AFP in clinical samples.


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ASSOCIATED CONTENT Supporting Information Experiment details. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author [email protected]

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This research was supported by the 973 Program (2013CB733700, 2014CB748500), National Natural Science Foundation of China (21125522, 21305045) and Fundamental Research Funds for the Central Universities (222201313004).

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SYNOPSIS TOC. A redox-mediated indirect fluorescence immunoassay (RMFIA) is reported here for the detection of the disease biomarker α-fetoprotein (AFP) using dopamine functionalized CdSe/ZnS quantum dots. This study makes the first use of redox-mediated fluorescence immunoassay for the rapid detection of AFP, opening up a new pathway for the detection of disease biomarkers.

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Redox-Mediated Indirect Fluorescence Immunoassay for the Detection

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