Magnetic Bead-Based Enzyme-Chromogenic Substrate System for

Apr 30, 2014 - Magnetic Bead-Based Enzyme-Chromogenic Substrate System for Ultrasensitive Colorimetric Immunoassay Accompanying Cascade Reaction for E...
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Magnetic Bead-Based Enzyme-Chromogenic Substrate System for Ultrasensitive Colorimetric Immunoassay Accompanying Cascade Reaction for Enzymatic Formation of Squaric Acid-Iron(III) Chelate Wenqiang Lai, Dianping Tang,* Junyang Zhuang, Guonan Chen, and Huanghao Yang* Key Laboratory of Analysis and Detection for Food Safety (Fujian Province & Ministry of Education of China), Department of Chemistry, Fuzhou University, Fuzhou 350108, P.R. China ABSTRACT: This work reports on a simple and feasible colorimetric immunoassay with signal amplification for sensitive determination of prostate-specific antigen (PSA, used as a model) at an ultralow concentration by using a new enzyme-chromogenic substrate system. We discovered that glucose oxidase (GOx), the enzyme broadly used in enzyme-linked immunosorbent assay (ELISA), has the ability to stimulate in situ formation of squaric acid (SQA)-iron(III) chelate. GOx-catalyzed oxidization of glucose leads to the formation of gluconic acid and hydrogen peroxide (H2O2). The latter can catalytically oxidize iron(II) to iron(III), which can rapidly (99 wt %), FeCl2·4H2O (>99 wt %), and glutaraldehyde (GA, 25 wt %) were obtained from Fuchen Chemicals (Tianjin, China). Colloidal gold (AuNP) with 16 nm diameter was synthesized according to our previous report.45 Magnetic beads (MB; particle size, ∼100 nm) in an aqueous suspension with a concentration of 25 mg mL−1 were obtained from Chemical GmbH (Berlin, Germany). All reagents (analytical grade) were used as received without further purification. Ultrapure water (18.2 MΩ cm) was purified by a Millipore-Q system. In the preparation of a carbonate buffer solution of pH 9.6, Na2CO3 (1.59 g), NaHCO3 (2.93 g), and NaN3 (0.2 g) were dissolved in 1000 mL of double-distilled water. Phosphate-buffered saline (PBS) solutions with various pH values were prepared by using Na2HPO4 and KH2PO4, and 0.1 M NaCl was used as the supporting electrolyte. Clinical serum samples were made available by Fujian Provincial Hospital, China. Preparation of mAb1-MB Conjugates. Monoclonal mAb1 antibody-conjugated magnetic bead (designated as MBmAb1) was prepared similar to our previous report.46−48 Before conjugation with mAb1, the magnetic bead was initially separated using an external magnet and then dried in the vacuum at 80 °C for 1 h. Following that, 50 mg of MB was added into 1 mL of anhydrous ethanol and the resulting mixture was sonicated for 10 min at room temperature (RT) to obtain a homogeneous suspension. Afterward, 30 μL of APTES (98 wt %) was injected in the mixture and continuously stirred for 6 h at RT. During this process, the aminated MB was formed based on the reaction between −OCH3 and −OH on the MB. The functional MB was separated and redispersed into 1 mL of PBS (pH 7.4) containing 300 μL of glutaraldehyde (25 wt %). The suspension was stirred vigorously for 6 h at RT. After magnetic separation, the precipitate was dissolved into 1 mL of carbonate buffer (pH 9.6) solution containing 100 μg of mAb1 antibody and shaken on a shaker (MS, IKA GmbH, Staufen, Germany) overnight at 4 °C. The excess mAb1 antibody was removed by magnetic separation. Subsequently, the mAb1-MB conjugates were treated with 10 wt % BSA-PBS (1.0 mL, pH 7.4) at 4 °C for 2 h to block the unreacted and nonspecific sites. Finally, 100 μL of sodium cyanoborohydride (25 mg mL−1) was injected into the suspension in order to reduce the resultant Schiff bases. The as-prepared MB-mAb1 conjugates were collected by using an external magnet and 5062

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Following that, 70 μL of 4 mM SQA was quickly injected into the resulting solution. After interaction for 1 min, the absorbance was recorded at λ = 468 nm on a Lab Microplate Reader (SH-1000, Corona Electric Co., Ltd., Japan). Colorimetric Immunoassay for Target PSA Using the SQA-Iron(III) System. Scheme 1b represents the immunoassay process toward target PSA by coupling with the SQAiron(III) system. The detailed procedure was summarized as follows: (i) 50 μL of PSA standards or samples with different concentrations and 25 μL of MB-mAb1 suspension (6 mg mL−1) were initially added into a 0.5 mL centrifuge tube, and the mixture was then incubated for 30 min at 37 °C on a shaker to form the antigen−antibody complex; (ii) 50 μL of GOxAuNP-pAb2 prepared above was injected into the centrifuge tube and incubated for another 30 min at 4 °C to form the sandwiched immunocomplex; (iii) 50 μL of glucose (4 mM) in pH 7.0 PBS was added into the centrifuge tube and incubated for 30 min at 37 °C on the shaker for enzymatic reaction; (iv) 50 μL of 10 mM HCl, 70 μL of 8 mM FeCl2, and 70 μL of 4 mM SQA were added to the centrifuge tube in turn; and (v) the absorbance was registered and recorded at λ = 468 nm on a plate reader after reaction for 1 min at RT (Note: The resulting mixture was separated with an external magnet and washed with pH 7.4 PBS after steps i and ii. The control tests with normal (negative) samples and the evaluations for clinical specimens were performed accordingly. All measurements were done at RT (25 ± 1.0 °C).

dispersed into 1 mL of PBS (pH 7.4) containing 1.0 wt % BSA and 0.1 wt % sodium azide. Preparation of GOx/pAb2-Conjugated Gold Nanoparticle (GOx-AuNP-pAb2). The GOx-AuNP-pAb2 conjugates were synthesized and prepared consulting to the literature with a little modification.47 Prior to labeling, the as-prepared gold colloids (AuNP, C[Au] ≈ 0.24 μM) with 16 nm in diameter were initially were adjusted to pH 9.0−9.5 by directly using 0.1 M Na2CO3 aqueous solution. With gentle stirring, the mixture containing 1.0 μL of pAb2 (1.0 mg mL−1) and 10 μL of GOx (1.0 mg mL−1) was added into 1.0 mL of colloidal gold. After incubation overnight at 4 °C, the mixture was centrifuged (14 000g) for 20 min at 4 °C. The pellet (i.e., GOx-AuNP-pAb2) was resuspended into 1.0 mL of sodium carbonate solution (2 mM) containing 1.0 wt % BSA and 0.1% sodium azide, pH 7.4, and stored at 4 °C until use. Monitoring of GOx Activity Using the SQA-Iron(III) System. Scheme 1a displays the assay mechanism of the SQAScheme 1. Schematic Illustration of (a) SQA-Stimulated Colorimetric Assay for Monitoring of GOx Activity and (b) SQA-Based Colorimetric Immunoassay by Coupling with the SQA-Iron(III) Systema

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RESULTS AND DISCUSSION Principle of SQA-Based Colorimetric Assay. In this work, monoclonal antihuman PSA antibody (mAb1) is immobilized on the MB by using glutaraldehyde as crosslinkage reagent (mAb1-MB), which is used as the immunosensing probe for the capture of target PSA. MB is not only used as a substrate for the conjugation of mAb1 antibody but also enables the rapid separation and purification of bionanocomposites after synthesis. Gold nanoparticles heavily functionalized with GOx and pAb2 antibody (GOx-AuNP-pAb2) is formed possibly owing to the dative binding between AuNP and free -SH of the proteins. The as-prepared GOx-AuNP-pAb2 is employed as the signal-transduction tag (detection antibody) for the construction of the SQA-based colorimetric immunoassay. In the presence of target PSA, the sandwiched immuneconjugates can be formed between MB-mAb1 and GOx-AuNPpAb2. Accompanying the GOx-AuNP-pAb2, the carried GOx can trigger the enzymatic catalytic reaction to produce the colored product. Initially, the GOx-biocatalyzed oxidization

a

MB, magnetic beads with 100 nm in diameter; AuNP, gold nanoparticle with 16 nm in diameter.

iron(III) system. Initially, 10 μL of GOx with the specified concentration (from 0 to 1000 μg mL−1) was added in 50 μL of PBS (0.5 mM, pH 7.0) containing 4 mM glucose. The resulting solution was incubated for 30 min at 37 °C. Then 70 μL of 8 mM FeCl2 dissolved into 50 μL of 10 mM HCl was added into the above-prepared mixture and reacted for 1 min at RT.

Figure 1. (A) UV−vis absorption spectra of (a) GOx + glucose + iron(II) + SQA, (b) GOx + iron(II) + SQA, (c) GOx + glucose + iron(II), (d) GOx + glucose + SQA, and (e) glucose + iron(II) + SQA, respectively, and (B) the corresponding photographs for part A. 5063

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Figure 2. (a) Effect of different reaction times between SQA and iron(III) [4 mM iron(III) used in this case] on the absorbance intensity of the SQA-iron(III) system, (b) comparison of the absorbance intensity of the SQA after interaction with different ions (Fe3+, Fe2+, Cu2+, Ni2+, Zn2+, Mn2+, Co2+, Ca2+, Mg2+, and Al3+, 1.0 mM for Fe3+ vs 20 mM for other ions), (c) the corresponding photographs for part b, and (d) calibration plots of the SQA system toward iron(III) standards with various final concentrations (inset: corresponding linear plots).

cause the change in the color of the SQA from the colorless to bluish red (photograph a in Figure 1B). Further, we also found that the absorbance increased with the increasing GOx concentration under the same conditions (please see Figure 3c). To further investigate the feasibility of the SQA-iron(III) system, we also investigated UV−vis absorption spectroscopy and the visible color of different components in the absence of GOx, glucose, iron(II), or SQA. For comparison, we initially monitored the mixture containing GOx, glucose, and iron(II) (i.e., in the absence of SQA). As seen from curve c in Figure 1A, no absorption peak was appeared at 468 nm. Meanwhile, the mixture was colorless (photograph c in Figure 1B). Favorably, when glucose (curve b and photograph b), iron(II) (curve d and photograph d), or GOx (curve e and photograph e) was absent in the detection system, the absorption peak and the color were almost the same as that of curve c and photograph c, respectively. The results revealed that (i) iron(II) could not cause the appearance of the absorption peak at 468 nm after incubation with SQA, (ii) transition of iron(II) to iron(III) could be triggered by GOx toward the catalytic reaction of glucose, (iii) the cascade reaction could be successfully carried out only in the simultaneous presence of glucose/GOx/iron(II)/SQA, and (iv) the visible color originated from the interaction between iron(III) and SQA not iron(II). Hence, we might suspect that when the GOx was conjugated onto the detection antibody, the SQA-iron(III) strategy could be employed for the development of the sandwiched immunoassay by monitoring the change in the absorbance or color. Evaluation and Characteristics of the SQA-Iron(III) System. In the SQA-iron(III) system, the cascade reaction mainly consisted of three steps: (i) the catalytic reaction of GOx toward the glucose, (ii) the redox reaction between hydrogen peroxide and iron(II), and (iii) the chelating reaction

toward the added glucose leads to the formation of gluconic acid and hydrogen peroxide (H2O2) with the participation of oxygen. The generated hydrogen peroxide can catalytically oxidize iron(II) to iron(III) in the acidic solution, which can be chelated with the SQA to form dimeric squarate complex. Formation of the iron-squarate complex causes the color of detection solution to change from bluish purple to bluish red accompanying the increasing absorbance with the increment of iron(III) concentration. The change in the color/absorbance indirectly depends on the concentration of target PSA in the sample. By monitoring the shift in the absorbance, we can quantitatively determine the concentration of target PSA in the sample. Vice verse, we can also qualitatively judge the PSA level by evaluating the change in the visible color. In contrast, GOxAuNP-pAb2 cannot be conjugated onto the functionalized mAb1-MB in the absence of target PSA; therefore, it can not trigger the progression of SQA-based colorimetric reaction. Control Tests for the SQA-Iron(III) System. For the successful development of SQA-based colorimetric immunoassay, one of the very important preconditions was whether the SQA-iron(III) system could be smoothly progressed in the presence of GOx, as depicted in Scheme 1a. To demonstrate this concern, several relative control tests were put into effect under the different conditions by using UV−vis absorption spectroscopy (Figure 1A) and the colorimetric measurement (Figure 1B) (Note: 10 μL of 1 mg mL−1 GOx, 50 μL of 1 mM glucose, 70 μL of 8 mM Fe2+, and 70 μL of 4 mM SQA were used in this case, respectively). As shown from curve a in Figure 1A, a strong absorbance peak at 468 nm was observed after the added GOx reacted with the mixture containing glucose, iron(II), and squaric acid. The reason might be most likely as a consequence of the fact that the generated iron(III) through the catalytic reaction of enzymatic product toward iron(II) was chelated with the SQA to form dimeric squarate complex. Moreover, the newly formed SQA-iron(III) chelates could 5064

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Figure 3. (a) Absorbance intensity of the SQA-iron(II)-H2O2 system toward H2O2 standards with various final concentrations (inset: corresponding linear plots), (b) effect of different catalytic times between GOx and glucose on the absorbance intensity of the SQA-iron(III) system, and (c) catalytic reactivity of the GOx with different concentrations in the SQA-iron(III) system.

shown in Figure 3a. Inspiringly, low-concentration H2O2 could also cause the change in the absorbance. When using 70 μL of 8 mM Fe2+ and 70 μL of 4 mM SQA as the detection solution, the system could allow the detection of H 2 O 2 at a concentration as low as 10 μM. The results further suggested that the presence of H2O2 in the iron(II)-SQA system could trigger formation of the iron(III)-SQA chelate, providing a feasibility for the SQA-based immunoassay. Following that, we also investigated the cascade reaction accompanying the SQA-iron(III) strategy, as illustrated in Scheme 1a. As described above, the reactions between H2O2 and iron(II) and between iron(III) and SQA were very fast. Toward bioactive GOx enzyme, however, the catalytic efficiency toward glucose relied on the catalytic time and temperature to some extent. Usually, the normal body temperature (37 °C) is suitable for enzymatic reaction. At this condition, we monitored the effect of different incubation times between GOx and glucose on the absorbance (Note: 10 μL of 1 mg mL−1 GOx, 50 μL of 2 mM glucose, 70 μL of 8 mM Fe2+, and 70 μL of 4 mM SQA were used in this case). As indicated from Figure 3b, the absorbance increased within the initial 30 min and then tended to slightly decrease. The reason might be attributed to the fact that H2O2 can be decomposed by light irradiation. Therefore, 30 min was selected for enzymatic reaction in this work. The enzymatic reactivity of GOx in the cascade reaction system was also studied. Various amounts of GOx were added to the system, and the absorption spectra were recorded. In this case, 50 μL of 4 mM glucose, 70 μL of 4 mM SQA, and 70 μL of 8 mM iron(II) were used for detection of 10 μL of GOx with various concentrations. As seen from Figure 3c, the absorbance increased with the increment of GOx concentration in the sample. At the low-concentration GOx, the absorbance exhibited a strong shift, and the detection limit was ∼1 μg mL−1 GOx. On the basis of these results, we might make a conclusion that the designed cascade reaction strategy based on the SQA-iron(III) system could be utilized for the determination of GOx by coupling with the change in the color or absorbance. Analytical Performance of Colorimetric Immunoassay Using the SQA-Iron(III) System. To further investigate the capability of the developed SQA-iron(III) system in the colorimetric immunoassay, the as-prepared GOx-AuNP-pAb2 was employed as secondary antibody for the detection of target PSA with a sandwich-type immunoassay format on the MBmAb1 by using the SQA-iron(III) system. Under the optimal

between iron(III) and SQA. For the development of the SQAiron(III) system, each step for cascade reaction should be investigated. Initially, the chelating reaction between iron(III) and SQA was monitored by using UV−vis absorption spectroscopy after interaction of the SQA (70 μL, 4 mM used in the case) with a different concentration of iron(III). To achieve an optimal detectable signal, the chelating time between SQA and iron(III) should be optimized (4 mM SQA used as an example). As indicated from Figure 2a, the absorbance rapidly increased with the increment of chelating time and tended to level off after 55 s. Longer chelating time did not cause the significant change in the absorbance. So, the colorimetric reaction was very fast. Compared with conventional HRPTMB-H2O2 system (∼600 s), the reaction between SQA and iron(III) was largely rapid (∼55 s). Significantly, the SQA toward iron(III) was specific and selectable. As shown from Figure 2b, a significant change in the absorbance was observed at 468 nm with the iron(III) against 20-fold higher interfering components (e.g., Fe2+, Cu2+, Ni2+, Zn2+, Mn2+, Co2+, Ca2+, Mg2+, and Al3+), which was almost in accordance with the photographs in Figure 2c. Although Cu2+ ion could cause the increase in the absorbance, the density was less. Meanwhile, the absorption peak triggered by Cu2+ ion was shifted to 494 nm. Hence, the SQA could be used for the detection of iron(III). To realize the subsequent SQA-based colorimetric immunoassay, the SQA system must have the ability to exactly differentiate the iron(III) with various concentrations. Next, we investigated the analytical performance of the SQA (70 μL of 4 mM SQA used in this case) toward iron(III) with various concentrations under the same conditions. As shown in Figure 2d, the absorbance increased with the increasing iron(III) concentration. Moreover, a linear range could be fitted in the working range of 14 μM to 1.12 mM, as shown from the inset in Figure 2d, and the detection limit could be lowered to 5 μM. The results revealed that the SQA system was feasible for quantitative monitoring of iron(III) in the sample. As is well-known, hydrogen peroxide (as s strong oxidizer) can oxidize iron(II) to iron(III). In this system, however, a puzzling question to be produced was whether the generated H2O2 could induce iron(II) to iron(III) for the formation of the iron(III)-SQA chelate. Therefore, the effect of different concentrations of H2O2 on the iron(II)-SQA system was also investigated by assaying the absorbance of the mixture at 468 nm after reaction with H2O2/iron(II)/SQA. Upon addition of H2O2 into the iron(II)-SQA system, the absorbance at 468 nm increased with the increasing H2O2 level in the solution, as 5065

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Figure 4. (a) Absorbance intensity and (b) calibration plots of the SQA-based colorimetric immunoassay by coupling with the SQA-iron(III) strategy toward different concentration PSA standards.

The results clearly indicated the high specificity of the SQAbased colorimetric immunoassay.

conditions, PSA standards with different concentrations were monitored based on the designed immunoassay protocol. The absorbance increased with the increment of target PSA concentration in the sample (Figure 4a). The curve is not a linear one, as is commonly observed for immunoassays. A curve-fitting procedure could be used for the calibration procedure. A pseudolinear relationship between the absorbance and the logarithm of PSA level, however, could be fitted to the experimental points from 1 pg mL−1 to 30 ng mL−1 (Figure 4b). The detection limit (LOD) was 0.5 pg mL−1 estimated at the 3sblank criterion, which was lower than the threshold value of total PSA in normal human serum (∼4 ng mL−1)49 and commercialized PSA ELISA kits (e.g., 0.5 ng mL−1, Biocell Biotechnol. Co., Ltd., Zhengzhou, China; 8 pg mL−1, Abcam Ltd., HK; 0.5 ng mL−1, Diagnostic Automation/Cortez Diagnostics, Inc. USA; 8 pg mL−1, Sigma-Aldrich, catalog no. RAB0331). We suspect that improvement of the sensitivity might be attributed to highly efficient enzyme-chromogenic substrate system as a result of the strong reaction between iron(III) and SQA. More favorably, the SQA-based colorimetric immunoassay could execute all the steps within 1.5 h for one sample, which was obviously less than that of the commercialized human PSA ELISA kit (∼3 h). The reproducibility and precision of the SQA-based colorimetric immunoassay were evaluated by calculating the intra- and interbatch variation coefficients (CVs). Results revealed that the CVs of the intra-assay with this method were between 4.3% and 7.8% (n = 5) in all cases. The batch-to-batch reproducibility of the SQA-based colorimetric immunoassay was also monitored by assaying 5 ng mL−1 PSA (as an example) for six times within the different days, and the obtained CV was about 8.9%. The low CVs might be attributed to the specific antigen−antibody reaction and the interaction between iron(III) and SQA. Thus, the precision and reproducibility of the SQA-based colorimetric immunoassay were acceptable. Further, the specificity of the SQA-based colorimetric immunoassay was also investigated toward other possible interfering substances, such as alpha-fetoprotein (AFP), cancer antigen 125 (CA 125), carcinoembryonic antigen (CEA), and human immunoglobulin G (HIgG). The reason for the use of these samples is that they usually coexist in the normal human serum. The comparative study was performed by measuring the low-concentration target PSA and high-concentration interfering components. As shown from Figure 5, higher absorbance was acquired with target PSA than those of other components.

Figure 5. Specificity of the SQA-based colorimetric immunoassay against PSA (0.1 ng mL−1), AFP (10 ng mL−1), CEA (10 ng mL−1), CA 125 (10 U mL−1), and HIgG (10 ng mL−1).

Screening of Real Samples and Interlaboratory Validation. The measurement trueness and applicability of the SQA-based colorimetric immunoassay for testing clinical serum samples were evaluated by using the SQA-based sensing system and commercialized human PSA ELISA kit (SigmaAldrich, catalog no. RAB0331) as a reference method (Note: Sample preparation and ELISA procedure for 12 human serum specimens were described in detail in our recent work).11 The levels of PSA in these samples were calculated according to the mentioned-above calibration curve (i.e., Figure 4) when using the SQA-based colorimetric immunoassay. The assay results by using these two methods are summarized in Table 1. As indicated in Table 1, all the experimental values of t (texp) for 12 clinical serum samples were lower than the critical value of t (tcrit, tcrit[4, 0.05] = 2.77). The regression equation (linear) for these data could be fitted as follows: y = 1.038x − 0.4068 (R2 = 0.995, n = 30) (x-axis, by the colorimetric immunoassay; y-axis, by commercialized human PSA ELISA kit). The results revealed that the SQA-based colorimetric immunoassay could be utilized for the detection of target PSA detection in human serum in the clinical laboratory. 5066

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Table 1. Comparison of ELISA and the SQA-Based Colorimetric Immunoassay for the Detection of PSA in 12 Human Serum Specimens method; concentration [mean ± SD (RSD, %), n = 3, ng mL−1] sample no.a

found by the colorimetric immunoassay

found by ELISA

texp

± ± ± ± ± ± ± ± ± ± ±

1.34 ± 0.13 2.41 ± 0.1 4.63 ± 0.1 5.87 ± 0.09 2.77 ± 0.08 3.51 ± 0.04 16.1 ± 0.76 24.3 ± 0.9 9.75 ± 0.71 7.87 ± 0.43 no application no application

0.58 1.09 0.75 0.77 1.23 1.68 1.09 1.78 1.45 1.11 no application no application

1 2 3 4 5 6 7 8 9 10 11

1.45 2.58 4.48 6.12 2.61 3.68 17.0 22.5 10.71 8.46 0.582

0.3 0.25 0.33 0.55 0.21 0.17 1.2 1.5 0.9 0.81 0.029

12

0.091 ± 0.016

a

Samples 1−6 were clinical serum specimens, and samples 7−12 were the diluted samples by using newborn cattle serum.

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CONCLUSIONS In this work, we for the first time demonstrate the ability of unconventional ELISA product for exceptional application in the colorimetric immunoassay based on enzymatic formation of the squaric acid-iron(III) chelate. The signal was amplified through an enzyme-catalyzed cascade reaction. Experimental results indicated that the visible color of the SQA-iron(III) system could be successfully triggered by the catalytic product of GOx toward glucose. Compared with traditional enzymebased colorimetric immunoassay, the SQA-based colorimetric immunoassay was sensitive, rapid, and feasible. Moreover, the SQA-iron(III) system is not susceptible to interference and changes in the assay conditions during the color generation stage. Importantly, magnetic bead-based colorimetric immunoassay can be suitable for use in the mass production of miniaturized lab-on-a-chip devices and open a new opportunity for protein diagnostic and biosecurity.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +86-591-2286 6125. Fax: +86-591-2286 6135. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National “973” Basic Research Program of China (Grant 2010CB732403), the National Natural Science Foundation of China (Grants 41176079, 41076059, and 21125524), the National Science Foundation of Fujian Province (Grant 2011J06003), and the Program for Changjiang Scholars and Innovative Research Team in University (Grant IRT1116).

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REFERENCES

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Analytical Chemistry

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dx.doi.org/10.1021/ac500738a | Anal. Chem. 2014, 86, 5061−5068