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Article Cite This: ACS Omega 2019, 4, 5083−5087
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Signal-Enhanced Lateral Flow Immunoassay with Dual Gold Nanoparticle Conjugates for the Detection of Hepatitis B Surface Antigen Youming Shen† and Guangyu Shen*,‡ †
ACS Omega 2019.4:5083-5087. Downloaded from pubs.acs.org by 31.40.211.195 on 03/09/19. For personal use only.
Hunan Province Cooperative Innovation Center for The Construction & Development of Dongting Lake Ecological Economic Zone and ‡College of Chemistry and Material Engineering, Hunan University of Arts and Science, Changde 415000, P. R. China ABSTRACT: We developed a signal-enhanced lateral flow immunoassay based on dual gold nanoparticle conjugates for the determination of hepatitis B surface antigen, in which no additional operation step for signal amplification was required. The first conjugates were gold nanoparticles modified with biotin and antibody. The second conjugates were gold nanoparticles modified with streptavidin. They were immobilized on different conjugate pads. When the sample solution flowed along the strip, the first conjugates were captured on the test line via antigen−antibody reaction, while the second conjugates were also immobilized on the test line through biotin− streptavidin. The signals were enhanced due to twice aggregation of gold nanoparticle conjugates. The immunoassay was used to detect hepatitis B surface antigen in the range of 0.1−30 ng/mL. The detection limit is 0.06 ng/ mL (based on S/N = 3). The method was simple, rapid, quantitative, and onsite. The application can provide a new platform for detection of hepatitis B surface antigen and even cancer markers. development of highly sensitive LFIA.19 However, fluorescence measurements require expensive device and fluorescent dyes are prone to photobleaching. To increase the sensitivity, Yang reported a cross-flow LFIA strategy based on the secondary coloring system of silver deposition.20 The silver deposition resulted in the enhanced color of the test line. But an additional step for silver deposition needs to be carried out, thus resulting in tedious operation. To overcome these shortcomings, another method for enhancement of the detection sensitivity was reported using a dendrimer-based gold nanoparticle cluster instead of the conventional gold nanoparticles.21 With this notion in mind, introducing secondary gold particles to bind with the primary gold particles accumulated on test line via the recognition between biotin and streptavidin, we attempted to develop a novel signal-enhanced LFIA for the detection of HBsAg in one step. Owing to the signal amplification using dual gold nanoparticle conjugates, the LFIA exhibits a low detection limit of 0.06 ng/ mL. The sensitivity of this LFIA is significantly higher than that of the previously reported luminescent quantum dot beadbased LFIA for HBsAg (0.075 ng/mL).22
1. INTRODUCTION Hepatitis B surface antigen (HBsAg) is one of the markers of virus replication in serum. The concentration of HBsAg provided basis for diagnosis and prognosis of cirrhosis and liver cancer caused by hepatitis B virus infection.1,2 Thus, accurate detection of HBsAg is of important significance in clinical diagnosis. Recently, some diagnostic methods, such as electrochemical immunoassay,3 chemiluminescence immunoassay,4 and enzyme-linked immunosorbent assay (ELISA),5 have been developed for the detection of HBsAg. Although these methods have some advantages, such as high sensitivity and accuracy, they are inconvenient for on-site detection due to their long incubation time, complex washing procedures, and heavy and large equipment. Compared to the above-mentioned methods, the lateral flow immunoassay (LFIA) is a simpler, faster, and more costeffective method.6 The method is based on the capillary action of fluid and colored nanoparticles, including colloidal gold,7 colloidal carbon,8 quantum dot,9,10 etc., used as colorimetric labels. Because red gold nanoparticles (GNPs) hold bright color, their size is easy to control, and are stable and biocompatible,11 LFIA has been widely applied in food safety,12,13 clinical laboratory test items,14,15 and environment monitoring.16,17 However, standard LFIA for HBsAg detection based on gold nanoparticles is always limited by its relatively low sensitivity (detection limit, 0.7 ng/mL).18 Much research has focused on improving sensitivity by introducing new labels or secondary coloring system. For example, Lee and co-workers used fluorescent nanoparticles and quantum dots as photoluminescent labels for the © 2019 American Chemical Society
2. EXPERIMENTAL SECTION 2.1. Reagents and Apparatus. The HBsAg and antiHBsAg antibodies were obtained from Shanghai Linc-Bio Science Co., Ltd. (Shanghai, China). Bovine serum albumin Received: December 21, 2018 Accepted: January 30, 2019 Published: March 8, 2019 5083
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adhesive backing layer. Each part overlapped 2 mm to ensure the solution migrating through the LFIA strip during the assay. 2.5. Assay Procedure. Sample solutions were prepared by adding HBsAg into running buffer (PBS + 0.1% Tween 20 (PBST) buffer) and placed into a 1 mL tube. Then, the sample pad was immersed in running buffer. The liquid migrated toward the absorption pad. After 5 min, red color development was observed at the test and control lines. To ensure that all antigens migrated to the test line, another 40 μL of running buffer was added to the tube. The intensity of the red color at test zone is proportional to the concentration of HBsAg. The red color was always observed at the control line, indicating that the performance of the strip was valid. After 10 min (total time of buffer running, which is defined as detection time), the intensity of the color at the test and control lines is evaluated using the “strip reader”.
(BSA) was obtained from Beijing Dingguo Biotechnology Company (Beijing, China). Biotin was purchased from the Biosharp Company (Hefei, China). Sodium chloride−sodium citrate (SSC), buffer 20× concentrate (pH 7.0), Triton X-100, Tris−HCl, HAuCl4, and sodium citrate were purchased from Sigma-Aldrich. Gold nanoparticles coated by streptavidin (SA−GNP) were obtained from Shanghai Bo Yao Biological Reagent Co., Ltd. (Shanghai, China). Cellulose fiber sample pads (JN8025) and glass fibers (GF-06) were obtained from Jiening Biological Technology & Co. Ltd. (Shanghai, China). Nitrocellulose (NC) membrane (HFB18002) was purchased from Millipore (Billerica, MA). Phosphate-buffered saline (PBS, 0.1 M, pH 7.0) was prepared using Na2HPO4 and NaH2PO4. Biojet BJQ 3000 dispenser, Airjet AJQ 3000 dispenser, guillotine cutting module CM 4000, and clamshell laminator were purchased from Biodot Ltd. (Irvine, CA). Strip reader, DT1030, was purchased from Shanghai Goldbio Tech. Co., Ltd. (Shanghai, China). 2.2. Preparation of Gold Nanoparticles (GNPs). Gold nanoparticles (16 nm) were synthesized according to the reported literature.23 Typically, a 0.01% HAuCl4 aqueous solution (250 mL) was added into a 500 mL round-bottom flask and then heated to boiling under vigorous stirring. Then, 1% sodium citrate (4.5 mL) was quickly added to the above solution. A color change of the solution from blue to red was observed. After that, the solution was cooled to room temperature under continuous stirring. It was stored in a refrigerator at 4 °C when not in use. 2.3. Conjugation of GNPs with Biotin and Antibody (Bio−GNP−Antibody). According to the paper reported by Wang and co-workers,24 Bio−GNP−antibody conjugates were prepared with slight modification. Before preparing, the gold nanoparticle solution was adjusted to pH 8.5 with 0.1 M K2CO3; 0.5 mL of 100 μg/mL biotin and 0.5 mL of 100 μg/ mL antibody solutions were added dropwise to 5 mL of a 5fold-concentrated GNP colloid under magnetic stirring. After stirring for 1 h, 0.5 mL of BSA (0.25%) was added into the solution to block the possible remaining active sites of the surface of gold nanoparticles. The mixture was incubated for 1 h at room temperature and centrifuged for 15 min at 12 000 rpm. The pellets were washed three times with PBS and redispersed in 5 mL of PBS at 4 °C for further use. 2.4. Preparation of LFIA Strip. Generally, conventional LFIA strip is composed of four parts: nitrocellulose (NC) membrane, sample pad, conjugate pad, and absorbent pad. However, the LFIA strip used in this work has two conjugate pads (SA−GNP conjugate pad and Bio−GNP−Ab conjugate pad). The sample pad was prepared by soaking in buffer (pH 8.0) containing 0.25% Triton X-100, 0.05 M Tris−HCl, and 0.15 mM NaCl. Then, it was dried and stored in desiccators at room temperature. Conjugate pads 1 and 2 were prepared by dispensing 4 μL of SA−GNP and 6 μL of Bio−GNP−Ab solution onto the different glass fiber pad with the dispenser Airjet AJQ 3000. The pads were dried at room temperature and stored in a desiccator at 4 °C. The test line and control line were prepared by dispensing capture antibody (1 mg/mL) and secondary antibody (1 mg/mL) solutions in different zones on the NC membrane with the dispenser Biojet BJQ 3000. When the membrane was dried for 1 h at room temperature, it was stored in a refrigerator at 4 °C. Finally, all pads and the NC membrane were assembled on a plastic
3. RESULTS AND DISCUSSION 3.1. Development of LFIA for HBsAg. The development of LFIA is based on dual gold nanoparticle conjugate as a colored reagent. The principle of LFIA for the determination of HBsAg is described in Figure 1. The strip used in this work
Figure 1. Principle of LFIA based on dual gold nanoparticle conjugates.
has two conjugate pads (conjugate pad 1 and conjugate pad 2). After the strip was inserted into the sample solution, the solution migrated toward the absorption pad and passed through conjugate pad 2, conjugate pad 1, test zone, and control zone. When the solution arrived at pad 2, the SA− GNP conjugates moved with the sample solution. When the solution arrived at pad 1, specific biological reactions of antibody−antigen and streptavidin−biotin took place simultaneously. Thus, the complexes of SA−GNP/Bio−GNP− antibody/HBsAg formed. Then, the complexes continued to migrate and arrived at the test and control zones, where sandwich-type reaction took place. Red bands in the test and control zones could be observed due to the accumulation of gold nanoparticles. When the blank sample solution migrated, only one red band was observed in the control zone. According 5084
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Rapid detection is an important advantage of LFIA. The optimum detection time was investigated by measuring the S/ N ratio as a function of detection time (buffer running time). The signal increased rapidly and reached a maximum at 10 min, then keeping a platform within 20 min. In view of rapid detection, 10 min was selected as the detection time (Figure 3C). 3.3. Calibration Curves of FLIA. Under optimal experimental conditions, quantitative detection was carried out. The peak areas increased with increasing HBsAg concentration. Figure 4 shows that the calibration plot of the peak areas versus HBsAg concentration is linear from 0.1 to 30 ng/mL. The detection limit is of 0.06 ng/mL (based on S/N = 3).
to the optical intensities of the red bands in the test zone, we can detect the concentration of HBsAg. To improve the LFIA sensitivity, the second conjugate pad (SA−GNP pad) was added and therefore more gold nanoparticles were accumulated on the test line, resulting in improved sensitivity. Figure 2 indicates the obvious difference of color intensity of test lines resulting from the use of only GNP−Ab conjugate (strip a) and two conjugates of SA−GNP and Bio−GNP−Ab (strip b).
Figure 2. Detection of HBsAg (0 ng/mL) by dual gold nanoparticle conjugate-based LFIA (a), conventional GNP-based LFIA (10 ng/ mL, (b)), and dual gold nanoparticle conjugate-based LFIA (10 ng/ mL, (c)).
Figure 4. Resulting calibration curve of LFIA with different concentrations of HBsAg. The concentrations corresponding to dots (from left to right) are 0.05, 0.1, 1, 10, 20, and 30 ng/mL. The error bars represent standard deviation, n = 3.
3.2. Optimization of Assay Conditions. Types of buffer solution affected the sensitivity and reproducibility of LFIA. Four running buffers, including PBS, PBS + 0.1% Tween 20 (PBST), PBST + 1% BSA, and 20 × SSC + 1% BSA, were used for the optimization by comparing S/N ratio on detecting 10 ng/mL of HBsAg. Figure 3A shows that PBST buffer is the best choice. The conjugate amount of SA−GNP and Bio−GNP−Ab on the conjugate pad affects the color intensity on both test and control zones. Therefore, the volume of conjugate solution dropped on the conjugate pad was investigated. Figure 3B shows that the highest values of S/N ratio were obtained at 4 μL for SA−GNP and 6 μL for Bio−GNP−Ab.
Table 1 compares the proposed LIFA with other methods. These results showed that the proposed LFIA shows favorable analytical performance. As a comparison, the same samples of HBsAg were detected with standard FLIA. The linear range is from 3 to 40 ng/mL and the detection limit is 1.8 ng/mL (based on S/N = 3). The detection sensitivity increased about 30-fold. 3.4. Selectivity and Reproducibility. To determine the specificity of this method, the response of the LFIA toward HBsAg (10 ng/mL) was compared to the responses resulting from 50 ng/mL of nonspecific species such as BSA, prostatespecific antigen (PSA), carcinoembryonic antigen (CEA), and α-fetoprotein (AFP) under the same experimental conditions.
Figure 3. (A) Effect of the buffer type on the signal-to-noise ratio of LFIA. (B) Effect of the volume of two conjugates dropped onto the conjugate pad on the signal-to-noise ratio of LFIA. (C) Effect of buffer running time on the signal-to-noise ratio of LFIA. The concentration of HBsAg is 10 ng/mL. 5085
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Table 1. Comparison of the Proposed LFIA and Other Methods assay method potentiometry impedance spectroscopy cyclic voltammetry LFIA LFIA LFIA
linear range (ng/mL)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: +86-736-7186115.
detection limit (ng/mL)
ref
ORCID
4−960 2.6−153.6
1.9 1.3
25 26
Guangyu Shen: 0000-0003-1121-3098
0.5−650 4.8−75
0.1 0.075 0.7 0.06
27 18 22 this work
The authors declare no competing financial interest.
0.1−30
Notes
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ACKNOWLEDGMENTS This work was supported by Hunan Provincial Natural Science Foundation of China (2016JJ6105).
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As shown in Figure 5, only HBsAg induced significantly larger signal, while signals resulting from other nonspecific species
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Figure 5. Specificity of LFIA to blank solution, BSA, PSA, CEA, AFP, and HBsAg. The concentration of nonspecific species is 50 ng/mL, and the concentration of HBsAg is 10 ng/mL, n = 3.
approximated those of blank solution, indicating that no crossreaction was observed. The reproducibility was also investigated using 10 ng/mL HBsAg. The coefficients of variation obtained for inter- and intra-assays were 6.6 and 8.4% (n = 5), respectively. The reproducibility of the FLIA is acceptable. 3.5. Detection of HBsAg. The feasibility of the proposed immunoassay for HBsAg is important. Five serum samples from different patients were examined using FLIA and ELISA. The results presented in Table 2 showed that the consistency between the data obtained by FLIA and ELISA is satisfactory. Table 2. Real Sample Analysis and Comparison with ELISA Method (n = 3) sample
ELISA (ng/mL)
this method (ng/mL)
relative deviation (%)
1 2 3 4 5
1.7 5.8 13.3 7.8 2.6
1.6 6.2 14.6 8.1 2.4
−5.9 6.8 9.8 3.8 −7.7
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4. CONCLUSIONS A signal-enhanced LFIA method based on dual gold nanoparticle conjugates without an additional operation step for signal amplification was developed. The assay can be used to detect HBsAg in the range from 0.1 to 30 ng/mL. Compared to the conventional LFIA, the detection sensitivity increased about 30-fold. The method was simple, rapid, quantitative, and on-site. The application can provide a new platform for detection of HBsAg and other cancer biomarkers by using the corresponding specific antibody. 5086
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