Fluorometric enzyme immunosensing system based on a renewable immunoreaction platform for the detection of Schistosoma japonicum antibody

Talanta 62 (2004) 735–740

Fluorometric enzyme immunosensing system based on a renewable immunoreaction platform for the detection of Schistosoma japonicum antibody Fu-Chun Gong a , Lian-hui Tang b , Guo-Li Shen a , Ru-Qin Yu a,∗ a

State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, PR China b Xiangya Medical College, Central South University, Changsha 410008, PR China Received 9 June 2003; received in revised form 17 September 2003; accepted 17 September 2003

Abstract A fluoroimmunosensing device which was based on ferulic acid (FA)/horseradish peroxidase system for the detection of Schistosoma japonicum antibody (SjAb) has been developed. To circumvent the difficulty of regeneration of immunocomposite surface, a natural chitosan-epoxy resin matrix was used for the immobilization of SjAg. The surface of the immunocomposite layer reacted was easily regenerated by simple polishing. The renewed surface served as a platform for the competitive immuno-reaction of HRP-SjAb and SjAb with SjAg immobilized at the support body surface and for enzymatic reaction. A novel fluorescent substrate ferulic acid for HRP, which is relatively stable toward H2 O2 , has been adapted in the proposed fluorometric enzyme immunosensing system. FA can been catalyzed to produce a non-fluorescent species. The amount of HRP-SjAb bound to the aforementioned renewable surface layer, which is related to the content of SjAb in samples could be quantitized by measuring the decrease of fluorescence of FA induced by HRP-SjAb. The chitosan incorporated in matrix is favorable for the amplification of this sensing system due to the electrostatic reaction with FA. The proposed method showed a linear response ranging from 45 to 150 ng ml−1 , with an improved detection limit of 45 ng ml−1 . The method has been employed to determine SjAb in serum samples. © 2003 Elsevier B.V. All rights reserved. Keywords: Schistosoma japonicum; Fluoroimmunosensing; Chitosan-epoxy resin matrix

1. Introduction Schistosomaisis is one of a parasite disease still widely spreading and threatening the health of human beings in some parts of Asian, Africa and Latin America [1]. Schistosoma japonicum antibody (SjAb) assay is a routine method for diagnosis of this disease. Traditional techniques based on detecting the precipitin of antigen–antibody reaction, such as indirect hemagglutination (IH) [2,3], immunoelectrophoretic assays (IEA) [4,5], erythroagglutination test (EAT) and complement fixation test (CFT) could not need the requirement of clinical analysis due to their low sensitivity. Some recent approaches based on immuno-labeling techniques such as enzyme-linked immunosorbent assay (ELISA) [6,7], ra-

∗

Corresponding author. Fax: +86-731-8822782. E-mail address: [email protected] (R.-Q. Yu).

0039-9140/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.talanta.2003.09.023

dioimmunoassays (RIA) [8], indirect fluorescent antibody test (IFAT) [9] made it possible to improve the sensitivity for SjAb assay. Unfortunately, they are time-consuming or requiring highly qualified personnel, RIA may bring with radioactive contamination [10,11]. Recently, electrochemical and piezoelectric immunosensing methods for SjAb detection have been investigated [12,13]. Though the problem of insufficient sensitivity remained unsolved for these approaches. Searching new, sensitive, simple and quantitative methods for SjAb assay is of considerable interest. The introduction of enzyme-linked immunoassay in early seventies makes it feasible to improve the sensitivity for SjAb assay matching with radioactive isotopes. Inspired by the success of the ELISA method, the present authors tried to develop fluorometric enzyme immunosensing device using FA as a substrate for HRP for SjAb assay. The immunosensing support body provides a renewable surface layer containing SjAb absorbed and encapsulated by chitosan-epoxy

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resin matrix. This sensing layer serves as a platform for competitive immuno-reaction of HRP-SjAb and the analyte SjAb in the incubation solution with SjAg at the surface of the supported biocomposite. The key point of the fluorometric enzyme amplification is the use of an appropriate substrate for fluorescence measurement. Ferulic acid (FA; 4-hydroxy-3-methoxyphenylcinnamic acid) as a cinnamic acid derivative is a naturally occurring phenolic compound found in the plant cell wall acting as an in vivo substrate for the plant peroxidases. To our knowledge, this reagent which drew the researchers’ attention was mainly focused on the protection of the plant against insect, fungal, viral, and avian attack [14]. This dibasic acid exhibits an extended resonance stabilization of the phenolate anion, hence slightly increasing its acidity relative to phenol. The extended unsaturated ring system of FA can emit fluorescence. As a substrate of HRP, FA can been catalyzed to produce phenoxyl radicals. The radicals can link up to form dimers, trimers and higher oligomers turning into a nonfluorescent species [15]. An outstanding feature of this reagent is its relatively high stability toward H2 O2 in the absence of HRP. In this paper, FA is used as an efficient substrate for HRP-catalyzed reaction in SjAb assay. The amount of HRP-SjAb bound to the aforementioned renewable surface layer, which is related to the content of SjAb in samples could be quantitized by measuring the decrease of fluorescence of FA. Owing to the combination of chemical amplification of enzymatic reaction with fluorometric measurement, an improved detection limit and stability for SjAb assay comparing to electrochemical methods could be obtained. The proposed procedure has been applied to the determination of SjAb in serum samples with results in good agreement with those obtained by other methods.

A blocking buffer for incubation containing 0.1% of bovine serum albumin (BSA) (w/w) in 0.1 M l−1 Tris–HCl and 10−3 M l−1 EDTA at pH 7.5 was used. A 0.1 M l−1 Tris–HCl 0.1 M l−1 KCl buffer of pH 7.5 was employed as a washing solution. The substrate solution was FA–H2 O2 in pH 8.0 phosphate buffer solution.

2. Experimental

2.4. Immobilization of SjAg

2.1. Reagents

An appropriate amount (7 mg) of Schistosoma japonicum antigen (SjAg) and 10 mg of bovine serum albumin were dissolved in 1.5 ml of cold pH 7.0 PBS (4 ◦ C) and mixed thoroughly with 900 mg of chitosan powder. The resulting mixture was left to dry in a desiccator at 4 ◦ C. The dried powder with SjAg-BSA adsorbed was thoroughly mixed with the epoxy resin with a weight ratio of 2:3. The resulting paste was squeezed into the polyvinylidene chloride (PVC) tube of 6 mm diameter to a depth of 1 cm contacting with a screwing nut at the other end of the tube.

Horseradish peroxidase (HRP, EC 1.11.1.7), 4-hydroxy3-methoxycinnamic acid (ferulic acid) and chitosan were purchased from Sigma. Epoxy resin and H2 O2 were obtained from Shanghai Chemical Reagents (Shanghai). All solutions were prepared in doubly distilled deionized water. Most chemicals used were of reagent grade. A 32 kDa molecular antigen of Schistosoma japonicum from adult worm antigen (AWA) was isolated and purified to homogeneity according to the reported method [16]. The concentration of SjAg is 4.5 mg ml−1 . The SjAb used in calibration procedure was prepared by immunizing rabbits for 45 days with Schistosoma japonicum 2500 [9]. The antibody in the infected rabbit serum was isolated by precipitation from saturated ammonium sulfate solution and purified as described in literature [17]. The actual concentration of stock SjAb solution (4.5 mg l−1 ) was determined by using ELISA method [18].

2.2. Apparatus Fluorescence measurements were made on a HITACHI F4500 fluorescence spectrophotometer (Japan). A peristatic pump was used to generate flowing stream (Jiangsu Electrochemical Instruments, Jiangsu, China). A model CSS501 thermostat (Chongqing Instruments, Chongqing, China) was employed to control the incubating temperature. The evaluation of the effect of pH on the activity of HRP was carried out with MultiSpec-1501 (SHIMDZU, Japan). 2.3. Preparation of HRP-SjAb An appropriate amount of HRP (10 mg) was dissolved in 0.5 ml of 1% glutaraldehyde in pH 6.8 PBS and left to incubate 12 h at room temperature. The resulting solution was dialyzed against 0.01 M l−1 PBS 0.15 M l−1 NaCl solution of pH 7.2 overnight at 4 ◦ C. SjAb (5 mg) dissolved in a mixture of 1 ml of 0.15 M l−1 NaCl solution and 0.1 ml of 1 M l−1 carbonate buffer at pH 9.6 were combined with a dialyzed HRP-glutaradehyde solution, and then incubated for 24 h at 4 ◦ C. The resulting solution was dialyzed against 0.01 M l−1 phosphate buffer solution at pH 7.2. Further purification was conducted by gel filtration on Sephadex G-200 column to give the HRP-SjAb conjugate (0.48 g l−1 ) which was used as the stock working solution.

2.5. Renewal of supported biocomposite surface The surface of supported biocomposite surface can be regenerated by turning the nut to extrude 0.05 ␮m thick outer chitosan-epoxy resin matrix/biocomposite layer and polishing with an alumina paper (0.05 ␮m) wetted with water to obtain a smooth, shiny surface. The new surface was finally cleaned with doubly distilled water.

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catalyzes the reaction converting a part of FA into nonfluorescent species. The fluorescence signal was recorded again. The decease of fluorescence intensity is calculated.

3. Results and discussion 3.1. Quantitative basis

Fig. 1. Configuration of immunosensing device integrated to flow injection system: (1) doubly optical fiber; (2) nut; (3) flow cell body; (4) micro cellulose-paraffin matrix incorporated with SjAg; (5) PVC tube; (6) vessel.

2.6. Measurement system and procedure The fluoroimmunosensing device configuration is illustrated in Fig. 1. The analytical procedure is shown as Fig. 2. The first step involved a 30 min incubation in the buffer solution (0.1 M l−1 Tris–HCl 0.1% BSA, pH 7.5) which contains 100 ␮l of HRP-SjAb tracer (480 ␮g l−1 ) and different volumes of SjAb (analyte) to give a final volume of 1 ml. The incubated surface of biocomposite was then rinsed thoroughly in a washing buffer solution (0.1 M l−1 Tris–HCl–KCl, pH 7.5) and stored in the same solution prior to the fluorescence measurement. Next, the supported biocomposite was fixed onto the flow-through reaction cell in the path of flowing stream integrating into a fluoroimmunosensing device. A substrate solution containing 2.5 × 10−2 M l−1 FA and 5×10−2 M l−1 H2 O2 was pumped through the flow-through path and detector cell. The fluorescence of FA is recorded at excitation and emission wavelengths of 380 and 460 nm, respectively. The biocomposite support body incubated in sample solution is inserted into the flow-through reaction cell in the flowing stream path, where the HRP of the HRP–Ab–Ag complexes bound on the biocomposite surface

Ferulic acid, which acts in nature as an in vivo substrate for the plant peroxidases, is used in the present SjAb assay as the substrate for HRP-catalyzed reaction. During the later process the FA is oxidized to phenolic radicals, which in turn link up to form nonfluorescent species. In the incubation procedure of competitive binding immunoassay, the SjAb from the analytical sample and HRP-SjAb added would compete to bind on the supported biocomposite surface. The amount of HRP-SjAb bound on the biocomposite surface after incubation would be reversely proportional to the amount of SjAb in the analytical samples. The concentration of SjAb in samples (analyte) is consequently reversely proportional to the fluorescence decrease of FA solution. Fig. 3 shows the change of the fluorescence of 2.5 × 10−2 M l−1 FA and 5×10−2 M l−1 H2 O2 solution after addition of different amount of HRP-SjAb. It was observed that HRP-SjAb caused a decrease of fluorescence intensity in dependence of its concentration. One would expect a similar phenomenon by sweeping a FA–H2 O2 solution over the supported biocomposite surface with bound HRP-SjAb-SjAg. This serves as the quantitative basis of the proposed sensing system for SjAb assay. 3.2. Response characteristics of the fluoroimmunosensing device The prepared sensing system was examined with the SjAb containing solution. Fig. 4 represents the results of

Fig. 2. Schematic illustration of competitive immuno-reaction and enzymatic reaction on the supported biocomposite surface and regeneration of the surface of the immobilized biocomposite.

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3.3. Regeneration, stability and reproducibility of the sensing device

250 excitation

A B C D E

emission

200 150 100 50 0

300

350

400

450

500

550

600

Wavelength(nm) Fig. 3. Fluorescence decrease of FA obtained with the addition of different HRP-SjAb concentration in phosphate-citric acid buffer solution containing 2.5 × 10−2 M l−1 FA and 5 × 10−2 M l−1 H2 O2 at pH 8.0. Concentration of HRP-SjAb was (ng l−1 ): (A) 0; (B) 1.2 × 10−2 ; (C) 2.4 × 10−2 ; (D) 4.8 × 10−2 ; (E) 9.6 × 10−2 .

Fluorescence intensity(arb. unit)

the time-dependent fluorescence response obtained with a SjAg-modified support body incubated in 3 ml of solution containing 0.24 mg l−1 HRP-SjAb with addition of different concentration of SjAb followed by fluorimetry. It indicates that the competitive immuno-reaction of the immobilized SjAg with HRP-SjAb/SjAb can cause an obvious change of fluorescence reading and the decrease of fluorescence intensity is concentration-dependent. One can also notice that the sensing system can reach steady-state response within 2 min and the H2 O2 alone can not induce obvious modification of fluorescence reading. This advantage of relatively high stability of FA toward the H2 O2 alone, which is associated with high catalytic efficiency of HRP to FA, has been ultilized in the present assay.

250

A

A major obstacle of the practical use of immunosensing devices is the difficulty associated with the regeneration of the sensing surface because the binding force between antigen and antibody is relatively strong. The use of chitosan-epoxy resin matrix results in a considerable retention of the activity of the grafted SjAg and the surface of the biocomposite can easily be renewed by simply polishing. The reproducibility of the regenerated sensing surface was evaluated by fluorimetry and a RSD of 2.7% was obtained. It implies that an additional advantage of the surface regeneration procedure is the lower cost of the proposed analytical approach. The immunocomposite support bodies were stored in a refrigerator (4 ◦ C) with a retention of the activity of the immobilized SjAg for at least 2 months. The repeatability of the prepared biocomposite (stored for 4 weeks) was evaluated with ten fluorimetric measurements in the use of 48 ␮g l−1 of SjAb solution. A good repeatability with a standard deviation of 1.5% was obtained. The BSA is added to reduce the nonspecific absorption of HRP-SjAb onto the surface sites without SjAg. The natural chitosan-epoxy resin matrix is favorable for the substrate molecules smoothly reaching the sites of HRP-SjAb-SjAg complexes due to the electrostatic reaction with substrate FA. 3.4. Effect of reaction pH The fluorescence intensity of FA solution is pH dependent, moreover, the activity of HRP-SjAb and the binding stability of HRP-SjAb-SjAg complexes are also affected by pH. The influence of pH on the fluorescence signal of FA was investigated. Fig. 5 shows the dependence of the fluorescence signal of 2.5 × 10−2 M l−1 FA solution on pH. Fluorescence intensity increases with pH up to pH 8, then

200

B

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C 100

D 50

E

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Time(sec.) Fig. 4. Time current-dependent response obtained with the incubated biocomposite support in solutions containng different concentration of SjAb (analyte) followed by fluorimetry in 2.5×10−2 to 5×10−2 M l−1 solution (pH 8.0). Concentration of SjAb was (␮g l−1 ): (A) 0; (B) 1.2 × 10−2 ; (C) 3.6 × 10−2 ; (D) 4.8 × 10−2 ; (E) 6.0 × 10−2 .

Fluorescence intensity (arb.unit)

Fluorescence intensity(arb. unit)

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8

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pH Fig. 5. Effect of pH on fluorescence intensity of FA. Concentration of FA was 2.5 × 10−2 M l−1 .

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Decrease of fluorescence(arb. unit)

0.7 0.6

Absorbance

0.5 0.4 0.3 0.2 0.1 0.0 0

2

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8

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200 180 160 140 120 100 80 60 40 20 1.7

1.8

pH Fig. 6. Effect of pH on the activity of SjAb-HRP. Absorbance measured at 472 nm.

tend to stabilize. The relatively lower pH lowered below 5.3 cause a decrease of fluorescence intensity. One should keep in mind that FA is hardly soluble in acidic medium and the solution pH affects the equilibrium between ground and excited states of FA. The effect of pH on the catalysis of HRP-SjAb was also examined. As FA has maximum absorption only in ultraviolet region, and the product of enzymatic reaction shows an absorption peak at 472 nm. The absorption at 472 nm is taken to monitor the enzyme-catalyzed reaction of FA as shown in Fig. 6. For HRP-SjAb, the absorption increases with pH up to 8, then tends to decrease at higher pH values. It indicates that the optimal pH of catalysis is 8. It seems that a low pH might be unfavorable for the tight binding between HRP-SjAb and SjAg, which causes loss of HRP-SjAb when substrate solution sweeps over the biocomposite surface. A pH of 8 was employed in most experiments. 3.5. Optimization of experimental parameters The selection of the amount of HRP-SjAb by incubating the biocomposite with increasing amount of HRP-SjAb was carried out. The response increases with the increase of the amount of conjugate up to 100 ␮l of HRP-SjAb (48 ␮g l−1 ) in 3 ml of incubating solution and then tend to saturate. This is due to the fact that the number of the epitopes of the antigen in the biocomposite surface is limited. Consequently, 100 ␮l of HRP-SjAb solution added to 3 ml of final incubation solution was routinely employed for the assays. An experimental study shows that the optimum incubation temperature is 27 ◦ C with an incubation of 30 min.

1.9

2.0

2.1

2.2

2.3

LogCSjAb(ng/ml) Fig. 7. Calibration curve for SjAb determination obtained by the competitive immunoassay. Biocomposite incubated in 0.1 M Tris–HCl 1 mM EDTA buffer solution of pH 7.5 containing different amount of SjAb (analyte), 48 ␮g l−1 SjAb-HRP conjugate and 0.1% (v/v) BSA. Each point represents the mean ± S.D. of four determinations. Table 1 SjAb determination with the prepared sensing device in rabbit serum samples Samplea

Fluoroimmunosensing method (g l−1 )

1 2 3 4

5.6 8.7 11.2 13.6

(31 days) (44 days) (55 days) (280 days)

± ± ± ±

0.22b 0.34 0.37 0.28

Conventional ELISA method 5.5 8.6 11.7 14.1

a The infected degree of samples is expressed by days of infection by Schistosoma japonicum 2000. The serum samples were diluted 100 times. b Mean ± S.D. of four measurements.

tended between 45 and 150 ng ml−1 . The sensitivity of proposed system was about a 2% of decrease of fluorescence intensity per 10−6 M of HRP-SjAb from the slope near the origin of the calibration curve. The detection limit for this system is 45 ng ml−1 of SjAb solution (determined as three times of the R.S.D. of the measurement blank) showing a marked improvement compared to the detection limit of 0.36 ␮g ml−1 achieved with electrochemical assay using the same immunoreagent [19]. The SjAg immunosensing system was used to determine the infected serum samples of rabbits, which were obtained from Xiangya Medical College, Central South University, Changsha, China. The results are shown in Table 1. It indicates that the results obtained by the proposed procedure were in good agreement with those obtained by ELISA method. The proposed immunosensing procedure can be used to detect the SjAb in serum samples.

3.6. Measurement with immunosensing system The calibration curve for Sj antibody detection is shown in Fig. 7. A nearly linear dependence was observed between the fluorescence decrease and the log CSjAb in the initial incubation. The pseudo-linear detection range for assay ex-

4. Conclusions The proposed procedure offers several advantages over conventional immunoassys for Schistosoma japonicum anti-

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body detection since it is based on a compact biocomposite with a renewable reaction interface. The use of the novel fluorescent substrate (ferulic acid) demonstrates the feasibility to improve the sensitivity and stability with fluorometric finish. The proposed methodology is useful for the immunoassay of others immunoreagents.

Acknowledgements This work was supported by the National Natural Science Foundation of China (Grants Nos. 20975006 and 29975006) and the Foundation for Scientific Committee of Hunan province.

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