CHINESE JOURNAL OF ANALYTICAL CHEMISTRYVolume 37, Issue 2, February 2009 Online English edition of the Chinese language journal

Cite this article as: Chin J Anal Chem, 2009, 37(2), 275–278.

Received 7 May 2008; accepted 4 September 2008 * Corresponding author. Email: panmin@zju.edu.cn; Tel: +86 0571-87951090; Fax: +86 0571-87951676 This work was supported by the Key Projects in the National Science & Technology Pillar Program during the eleventh five-year plan period of China (No. 2006BAD30B03). Copyright © 2009, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved. DOI: 10.1016/S1872-2040(08)60087-0

RESEARCH PAPER

Preparation and Immunochromatographic Assay of Biological Probes with Fe Nanowires/Chitosan/Antibody YANG Hao, YANG Xiao-He, CHEN Yu-Quan, PAN MIN*State Specialized Laboratory of Biomedical Sensors, Department of Biomedical Engineering, Zhejiang University, Hangzhou 310027, China

Abstract: Fe nanowires/chitosan nano-composites were prepared and their capacity for adsorbing bovine serum albumin (BSA) was examined. The results show that when pH of buffer solution was set at 6.0, the maximum adsorption was 148.40 mg g–1. By crosslinking -human chorionic gonadotropin ( -HCG), biological probes with Fe nanowires/chitosan/antibody were prepared. immunochromatographic strips were manufactured, and different concentration HCG antigens were tested. The results indicate that the sensitivity is 10 IU l–1. Pregnancy can be detected by these immunochromatographic strips 2–3 days earlier than by the colloidal gold test strips in the market. Key Words: Nanowires; Immunochromatographic Assay; Immunosensor; Biological probes

1 Introduction

In the 1980s, immunochromatographic assay was built on immunofiltration assay. Today, owing to being fast, simple, and cheap, immunochromatographic assay is applied widely in pregnancy and tumour detection. However, it can only be used for qualitative detection. Especially when the result is close to the detection limit, other traditional methods must be adopted to diagnose. Thus, discovering new marker for improving sensitivity and realizing quantificational detection is important.

Based on anodic aluminum oxide (AAO) template, Fe nanowires were electrochemically deposited. After coating by chitosan, Fe nanowires/chitosan nanocomposites were prepared, and glutaraldehyde was used to activate the surface of chitosan. The capacity for adsorbing bovine serum albumin (BSA) was tested at different pH of buffer solution and different concentrations of BSA. The biological probes with Fe nanowires/chitosan/ -HCG were synthesized by cross- linking with -HCG antibody. Human chorionic gonadotropin (HCG) antigen was captured by -HCG antibody immobilized on the strips and the sandwich was formed with the biological probes. In this study, nano-properties of Fe

nanowires were used in the qualitative detection of HCG. This will ensure good support to quantificational detection by magnetic detecting device[1,2]. 2 Experimental 2.1 Reagents and materials

Aluminum foil (purity, 99.999%, thickness, 0.5 mm) was

obtained from Beijing Mengtai Technology and Development Center, China. Chitosan (deacetylation degree >90%) and BSA were obtained from Sinopharm Chemical Reagent Co, Ltd., China. Mouse anti-human -HCG (6.4 g l–1), mouse anti-human -HCG (9.6 g l–1), goat anti-mouse IgG (7.3 g l–1) and HCG antigen (10, 25 IU l–1) were obtained from Shanghai Genering Biotech Co, Ltd., China. The other reagents used were analytical reagents and were not purified before use. Deionized water was used in all experiments.

Testing instruments: ultraviolet spectrophotometer, CARY 100, was purchased from Varian, USA. Dispensing system, XYZ3000, was purchased from BioDot, USA. Superconducting quantum interference device (SQUID),

GUO Zhi-Qiang et al. / Chinese Journal of Analytical Chemistry, 2009, 37(2): 275–278

MPMS XL-5, was purchased from Quantum Design, USA. Field-emission scanning electron microscope (FSEM), SIRION-100, was purchased from FEI, USA. Transmission electron microscope (TEM), JEM-1200EX, was purchased from JEOL, Japan.

2.2 Methods 2.2.1 Preparation of Fe nanowires

After suitable cutting, electrochemical polishing, and

washing, aluminum foil was dipped in 0.3 M oxalic acid solution with ice bathing, and anode oxidized at 40 V DC for 1 h. It was then soaked in a mixture of phosphoric acid and chromic acid for 1 h, and the same anode oxidation was repeated. After decreasing the anodic oxidation voltage step by step, the AAO template was obtained.

The electrochemical deposition solution was made of FeSO4·7H2O (l00 g l–1), (NH4)2SO4 (15 g l–1), MgSO4 (30 g l–1), ascorbic acid (1 g l–1), and glycerin (2 ml l–1). AAO template was dipped in it and the pH was set at 3. The optimum condition of electrochemical deposition was 15 V 50 Hz AC for 5 min.

After removing the aluminum substrate of the template by saturation SnCl4, the AAO template was dissolved by NaOH solution. It was washed with deionized water and enriched by magnetism repeatedly until the pH was 7.0. After supersonic, equable solution of Fe nanowires were obtained. 2.2.2 Preparation of Fe nanowires/chitosan

0.01 g Fe nanowires, 2.0 ml liquid paraffin, and 0.1 ml

span-80 were added in a 1.0-ml chitosan/acetic acid solution (10 g l–1), supersonic dispersed for 30 min. 0.15 ml glutaraldehyde solution (25%) was added, and shaken slowly at room temperature for 4 h. It was washed with petroleum ether, acetone, and deionized water in turn and enriched by magnetism repeatedly. Then, it was dispersed in 10 ml phosphate buffer solution with pH 7.0, and conserved at 4 ºC.

2.2.3 Experiment of Fe nanowires/chitosan absorbing BSA

BSA with different quantities and buffer solutions with

different pH were added in Fe nanowires/chitosan solution, shaken slowly at room temperature for 30 min, and separated by magnetism. The BSA concentration of the supernatant was then measured. The method was as follows: measuring absorbance at 280 nm by ultraviolet spectrophotometer, calculating remanent BSA concentration according to standard curve, and then obtaining the adsorbing value of BSA.

2.2.4 Preparation of biological probes with Fe

nanowires/chitosan/ -HCG

The pH of Fe nanowires/chitosan solution was set at 7.2, -HCG antibody was added, and the concentration was

adjusted to 0.1 g l–1, and shaken slowly at room temperature for 24 h. Then, 1 ml BSA (5%) was added, and shaken slowly at room temperature for 6 h. It was washed with phosphate buffer solution and enriched by magnetism repeatedly. It was then dispersed in 5 ml phosphate buffer solution with pH 7.2, and conserved at 4 ºC.

2.2.5 Experiment of HCG immmunochromatographic strips

Materials of immunochromatographic strips: membrance is

cellulose nitrate, sample pad and conjugate pad are glass fiber, and absorbent pad is plant fiber.

After pretreatment of sample pad and conjugate pad, conjugate pad was soaked in Fe nanowires/chitosan/ -HCG solution, and then placed in dry closet for 12 h.

-HCG antibody was added in phosphate buffer solution with pH 7.4 and the concentration was adjusted to 1.8 g l–1, similarly, for IgG antibody. The test line with -HCG antibody and the control line with IgG antibody were dispensed on membrance by BioDot dispensing system, respectively, and then dried at 35 ºC for 2 h.

All materials were assembled as shown in Fig.1, cut into strips, and placed in dry closet for 6 h.

The morning urine of a pregnant woman and the HCG antigen standard solution were tested with strips respectively.

3 Results and discussion 3.1 Characterization of Fe nanowires

AAO template, in which Fe nanowires were

electrochemically deposited, was observed by FSEM, as shown in Fig.2. It can be seen that the nano-holes in AAO template have been filled obviously. Their diameter is 50 nm, and their density is 1.0 × 106 mm–2.

Fig.1 Structure of HCG immunochromatographic strips

Fig.2 FSEM of AAO template after electrochemical deposition of Fe nanowires

GUO Zhi-Qiang et al. / Chinese Journal of Analytical Chemistry, 2009, 37(2): 275–278

1

m m

C Cq Kq q

The magnetism of Fe nanowires was analyzed by SQUID, as shown in Fig.3. The coercive force with magnetization perpendicular to the plane of AAO template is 1205 Oe, and that parallel is 401 Oe. It indicates that Fe nanowires arrays have a preferential magnetic orientation along Fe nanowires axis. The magnetization direction is obvious uniaxial magnetic anisotropy because of Figure anisotropy of Fe nanowires.

Fe nanowires were observed by TEM, as shown in Fig.4. The diameter was 50 nm, and the length was 750 nm after supersonic. The diameter of the nanowires is equal to the diameter of nano-holes in the AAO template.

3.2 Mechanism of Fe nanowires/chitosan Through supersonic, Fe nanowires are dispersed equably in

the mixture of span-80 and liquid paraffin, and the water-in-oil microemulsion system is formed. After Fe nanowires’ absorption of chitosan, the glutaraldehyde added in reacts with it, that is to say, carbonyl of glutaraldehyde reacts with amidogen of chitosan to produce schiff bases (–C

N–). The reaction makes the spiral and puckered structure of chitosan molecule more stable and the chain of chitosan molecule more ordinal. This performs well for the covalent conjugation of protein with chitosan.

3.3 Experiment of Fe nanowires/chitosan absorbing

BSA 3.3.1 Effect of pH on the absorption of BSA

The effect of different pH on the absorption of BSA is

shown in Table 1. The results show that when the pH of the buffer solution is set at 6.0, the maximum adsorption is 148.40 mg g–1. When the pH of buffer solution is set to be larger or less than this value, the absorption of BSA will decrease obviously.

Fe nanowires/chitosan absorption of BSA is mainly owing to electrostatic interaction. It is because the pKa of amidogen of chitosan is about 6.5[3], and the isoelectric point of BSA is 5.0[4,5]. When the pH is set between 5.0 and 6.5, the chitosan has positive charge and the BSA has negative charge. Thus, there is strong electrostatic interaction to attract each other. Otherwise, hydrophobic interaction also affects the absorption. These interactions are related to the structure of BSA at this pH[6,7].

3.3.2 Kinetics of Fe nanowires/chitosan absorbing BSA The absorption of BSA can be described using Langmuir

equation: C is the equilibrium concentration in g l–1, q is the

equilibrium absorption of BSA in mg g–1; qm is the saturated

Fig.3 Hysteresis loops of Fe nanowires

Fig.4 TEM of Fe nanowires

Table 1 Diagram of pH on BSA adsorption pH 4 5 6 7 8

Adsorption 81 102 148 120 110

Fig.5 Linear representation of Langmuir equation of BSA

absorption of Fe nanowires/chitosan in mg g–1; K is the equilibrium constant in l g–1.

According to the data shown in Fig.5, the results of linear fitting indicate that qm is 133.96 mg g–1, and K is 3.708 l g–1. The results are close to the experimental value.

3.4 Experiment of HCG immmunochromatographic strips

The contrastive experiment of HCG immunochromato-

graphic strips with colloidal gold strips is shown in Fig.6. The red test line and the red control line in (A) show that

colloidal gold strips are active. The weak red test line and the red control line in (B) show that 25 IU l–1 is the detection limit of colloidal gold strips. The brown test line and the brown control line in (C) show that 10 IU l–1 is the detection limit of HCG immunochromatographic strips.

GUO Zhi-Qiang et al. / Chinese Journal of Analytical Chemistry, 2009, 37(2): 275–278

Fig.6 Contrast of HCG immunochromatographic strips with

colloidal gold strips (A) colloidal gold strip tested by urine of pregnant woman, (B) colloidal gold strip tested by 25 IU l–1 HCG antigen, (C) HCG immunochromatographic strip tested by 10 IU l–1 HCG antigen

The results show that HCG immunochromatographic strips

with Fe nanowires/chitosan/ -HCG biological probes are more sensitive than the colloidal gold strips in the market. In the early period of pregnancy, the concentration of HCG increases one time every 1.7–2 days. Thus, pregnancy can be detected 2–3 days earlier by HCG immunochromatographic strips than by colloidal gold strips.

Colloidal gold links with -HCG by physical absorption, which relies on non-specificity absorption between nanoparticles and antibody, and thus, it is unstable. Fe

nanowires/chitosan links with -HCG by covalence conjugation, which relies on covalence reaction between active radical and antibody, and therefore, it is more stable.

Nanowires are larger than nanoparticles in three- dimensional space, therefore, nanowires have more space to immobilize antibody, namely, more capacity to capture antigen. Furthermore, more space on nanowires benefits the specificity of immunoreaction[8]. However, this does not indicate that larger materials will be better, because very large materials will cause harm to the preparation of biological probes and immunochromatographic assay.

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