Anal Bioanal Chem (2014) 406:6637–6646 DOI 10.1007/s00216-014-8093-0
RESEARCH PAPER
Nano-gold capillary immunochromatographic assay for parvalbumin Shuyuan Du & Hong Lin & Jianxin Sui & Xiudan Wang & Limin Cao
Received: 17 June 2014 / Revised: 2 August 2014 / Accepted: 5 August 2014 / Published online: 29 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract A novel non-instrumental bioanalysis based on colloidal-gold immunochromatography in a modified glass capillary was developed and named capillary immunochromatographic assay (CICA). In this report, glass capillary was proposed as a support in immunochromatographic assay because of its excellent characteristics. Goat anti-rabbit IgG and parvalbumin (PV) were immobilized on the inner wall of the glass capillary as control zone and test zone, respectively. The CICA was constructed, and main variables for the performance were optimized. Using an important allergen of fish products (parvalbumin, PV) as the target, the analytical efficiency of the developed technique was investigated and the visual detection limit (VDL) and semi-quantitative limit of detection (LOD) were estimated to be 70 ng mL−1 and 40 ng mL−1, respectively. The coefficient of variation (CV) for the intra-assay and inter-assay was calculated for the PV concentration of 50 ng mL−1, and the entire operation, including sample preparation, was consistently performed in 30 min. The developed technique was implemented and validated with different foodstuffs, including Scophthalmus maximus (Linnaeus), surimi products, and livestock, confirming sufficient accuracy and precision of results and verifying the method to be efficacious. These results enabled us to propose CICA as a new and promising technique for simple, rapid, and on-site screening of PV in biological samples.
Electronic supplementary material The online version of this article (doi:10.1007/s00216-014-8093-0) contains supplementary material, which is available to authorized users. S. Du : H. Lin : J. Sui : X. Wang : L. Cao (*) Food Safety Laboratory, Ocean University of China, Qingdao 266003, China e-mail: [email protected]
Keywords Capillary . Capillary immunochromatographic assay . Colloidal gold . Parvalbumin
Introduction Recently a variety of immunoassays have been developed in the fields of food, environmental analysis, and clinical applications. Traditional techniques, including immunosensors and enzyme-linked immunosorbent assay (ELISA), usually have satisfactory sensitivity, accuracy, selectivity, and precision [1–3]. However, they are relatively complex, time consuming, and largely dependent on professional equipment and operating experience [4–7], meaning their cost and complexity limits their practical applications, especially in field detection. Hence, there is increasing attention focusing on investigating techniques, for example immunochromatographic assays, that can improve the simplicity by using new low-cost and high-performance materials. In comparison to ELISA and other immunoassays, such techniques have unique advantages [8–10]: the operation (after sample pretreatment) can be accomplished in less than 20 min, and the final results can be qualitatively detected by the naked eye without any need of equipment. Most of these techniques, for example lateral-flow colloidal-gold immunoassay (LFIA) and dot-immunogold filtration assay (DIGFA), were developed using nitrocellulose membrane (NCM) as the carrier material. The high proteinbinding capacity of NCM may facilitate the immobilization of antibodies. However, NCM is subject to breakage, compression, and scoring during processing, and is also greatly affected by environmental conditions including temperature and relative humidity [11]. Towing phenomena, diffusion phenomena, and significant deviation (inter and intra) always exist in chromatography because of the complicated and heterogeneous structure of NCM. For these reasons, NCM-based immunochromatographic assays have relatively poor
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sensitivity, reproducibility, and repeatability, which limits their use in real applications. Other materials have recently been used in immunochromatographic assays as alternatives to NCM, including paper [12–15], thread [16, 17], and an SPE column [18]. Although these new techniques still seem far from commercial application, they provide very valuable ideas regarding development of more reliable, convenient, and efficient immunochromatographic techniques. In this study, a new strategy is proposed for the design and fabrication of an immunochromatographic assay based on a glass capillary. This is mainly motivated by the unique advantages of the glass capillary in comparison to other materials: low cost, less sample usage, easily modified, and favorable optical properties. More importantly, the uniform smooth surface and the high rigidity and ionic strength tolerance of the glass matrix make it more robust than flexible materials regarding possible interference from the environment and the sample matrix, which can be very helpful for improving the accuracy and precision of the analytical operations. Herein, a nano-gold capillary immunochromatographic assay (CICA) emulating the lateral-flow format was designed and fabricated, the main variables affecting the CICA response were investigated, and the performing conditions were optimized. Using an allergen (parvalbumin) in fish products as the target, the real efficiency of the technique for analyzing biological samples was validated, and its potential use as a new screening tool was also evaluated and discussed.
Materials and methods Reagents Parvalbumin and anti-parvalbumin antibody with an affinity constant of 1.58×109 L mol−1 were prepared and characterized as in our previous work [19–22]. Goat anti-rabbit IgG was purchased from Zhongshan Jinqiao Co. (Beijing, China). 3-Glycidyloxypropyltrimethoxysilane (98 %, GPTMS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrogen tetrachloroaurate (III) (HAuCl4), trisodium citrate, toluene, acetone, and triethylamine (TEA) were obtained from National Chemical Pharmaceutical Co. (Shanghai, China). Bovine serum albumin (BSA), human serum albumin, egg albumin, tris(hydroxymethylamino) metane (Tris), and Tween 20 were purchased from Solarbio (Beijing, China). Molecularweight markers were purchased from Fermantas Co. Ltd (Vilnius, Lithuania). All chemicals were of analytical grade unless otherwise stated. Apparatus The glass capillary (d=0.9 mm) was purchased from the instrument factory of West China University of Medical
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Sciences (Chengdu, China). The protein A column was purchased from GE Healthcare (GE, USA). The UV–vis spectra were recorded with a UV-1101 from Techcomp (Shanghai, China). The AuNPs and AuNP-labeled anti-PV conjugates were characterized by transmission electron microscope (TEM, JEOL JEM-1200EX, Japan). All centrifugations were performed with Z36HK (HERMLE Labortechnik GmbH, Germany). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results were recorded on a Tanon180 Gel imaging system (Tanon Science & Technology Co. Ltd. Shanghai, P. R. China). After immunochromatographic reaction, the images were scanned by an HP Scanjet G4050 scanner from Seiko Epson Nagano (HP, Japan) to obtain the brightness of the control and test zones. All aqueous solutions were prepared in ultra-pure water of 18.2 MΩ purified by Unique-R20 from Research Scientific Instruments Co. (Beijing, China). General principle The CICA system was constructed using the principle of direct competitive immunoassay. The PV and goat antirabbit IgG were immobilized on the inner wall of the glass capillary by covalent bonding to form the test zone (one end of capillary) and control zone (other end of capillary), respectively (Fig. 1a). The capillary was placed horizontally on a table, and a mixture of AuNP-labeled anti-PV and analyte was injected by a pipettor into the modified capillary to fully cover the test zone, and reacted for 4 min. Then the solution was forced to the control zone by the pipettor, and reacted for another 4 min. The unbound conjugate was removed from the capillary by pipettor, and the capillary was washed thoroughly by moving up and down in phosphate-buffered saline with Tween 20 (PBST, 1 g L−1 Tween 20 in 0.01 mol L−1 PBS, pH 7.4). For negative samples, two red bands appeared as a result of the accumulation of red AuNP-labeled anti-PV conjugate at both zones, and their brightness was almost the same (Fig. 1b) [23]. In contrast, for the positive samples the brightness of the test zone was visually weaker than that of the control zone, sometimes to the extent that no significant red band appeared (Fig. 1c). Preparation of AuNPs and AuNP-labeled anti-parvalbumin antibody AuNPs were prepared according to the literature, with slight modifications [24]. The size of AuNPs was controlled by subtle variation of the ratio of HAuCl4 to sodium citrate in the mixture. When the color of the solution stabilized, boiling continued for another 15 min. The mixture was heated under reflux to room temperature, and then stored at 4 °C in a dark bottle until further use. The AuNP-labeled anti-PV conjugate was prepared according to [25]. The crude anti-PV was purified by a protein
Nano-gold capillary immunochromatographic assay for parvalbumin Fig. 1 The scheme of the CICA system: (a) the control zone and test zone on the capillary, (b) negative results and (c) positive results
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b
a
c
Control
Control
Control
zone
zone
zone
antigen gold nanoparticle (AuNP)
Background AuNP-labeled antibody
Test
Test
Test
zone
zone
zone
Negative test
A column according to the instructions, and then centrifuged at 3000g to remove the precipitation. The optimum pH and concentration ratio of antibody to AuNP were determined by the Slot method [26]. The AuNPs were adjusted to the optimum pH with K2CO3 (0.1 mol L−1), and the anti-PV antibody was slowly added to the AuNPs with continuous stirring. The mixture was stirred gently for 2 h, and subsequently 10 % BSA and 1 % polyethylene glycol (PEG)-20000 were added to reach the final concentrations of 1.0 % (w/v) and 10 % (v/v), respectively. Then the mixture was reacted at room temperature for 30 min to block the residual surface of AuNPs, followed by centrifugation at 2500g for 15 min at 4 °C. The supernatant was further centrifuged at 9000–11,000g for 1 h at 4 °C. Finally, the precipitation was suspended in Tris–HCl (0.01 mol L−1, pH 8.2, containing 1 % BSA and 0.02 % NaN3) and stored at 4 °C until further use. The formation of AuNPs and AuNP-labeled anti-PV conjugate was checked with UV– vis spectra and TEM in Medical College of Qingdao University. For characterization of AuNP-labeled anti-PV conjugate, an indirect ELISA was performed as described in [27]. The variables were first optimized by checkerboard titrations [28], and the concentrations of PV and the HRP-labeled antibody were optimized as 10 μg mL−1 and 1.6 μg mL−1, respectively. Pretreatment and modification of the capillary The pretreatment and modification of the capillary were performed by the procedure described in [29], with modification. The glass capillary was immersed in a mixture of “piranha solution” (H2SO4:30 % H2O2 =3:1, below 80 °C) and sonicated for 15 min. Then it was rinsed thoroughly with deionized water until the pH of the cleansing solution was close to 7.0, and dried at 105 °C for approximately 60 min. The capillary was
goat anti-rabbit IgG
Positive test
then successively washed with KOH (1 mol L−1), water, HCl (1 mol L−1), water, and acetone, each for 15 min. Finally the capillary was dried at 105 °C for more than 1 h. After cleaning the capillary was modified immediately by the following procedure. It was first immersed in GPTMS solution (10 %, v/v in dry acetone, containing 1 % triethylamine) for 18 h at room temperature. Then the GPTMS solution in the beaker was removed and the reaction was continued for another 2 h. The capillary was washed thoroughly by moving up and down in toluene and acetone for 5 min, and dried by flushing with nitrogen. Then 3 μL PV was injected into one end of the capillary by pipettor to fabricate the test zone. The capillary was placed vertically in a well of ELISA plate at 25 °C for 2 h to immobilize antigen and fabricate the test zone on the inner wall. Redundant PV was removed from the capillary by pipettor, and the modified capillary was washed by immersing in PBST and moving up and down for 5 min. 3 μL goat anti-rabbit IgG was injected into the control-zone end of the capillary by pipettor. The control zone was fabricated on the inside wall of the capillary using the same procedure as for the test zone. After removing redundant goat anti-rabbit IgG and washing with PBST, the modified capillary was immersed in 1.5 % BSA for 2 h at 37 °C to block unspecific sites. Finally, the capillary was rinsed by PBST again as described above and stored at 4 °C until use. For optimization of the GPTMS modification time (in the range 4–48 h), the concentrations of PV and goat anti-rabbit IgG were 0.4 mg mL−1, and the color development time was 8 min. For optimization of the concentration of PV and goat anti-rabbit IgG for immobilization on the capillary, the GPTMS modification time was 18 h, the color development time was 8 min, and PBS was used for the blank. For
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Sample preparation Scophthalmus maximus (Linnaeus), surimi products, and livestock and poultry meat samples were collected from a local supermarket (Qingdao, China). The pretreatment of samples used a method described elsewhere, with modifications [30]. First, the samples were homogenized with Tris–HCl (10 mmol L−1, pH 7.5) at a ratio of 1:2 (w/v), followed by heating in a water bath at 98 °C for 5 min. Then the mixture was centrifuged at 3800g for 5 min and the supernatant was collected. The protein content of the extract was investigated by discontinuous SDS-PAGE under denatured conditions using a procedure described in [31], and the results were recorded using an HP Scanjet G4050 (Hewlett-Packard). The protein content of the prepared samples was determined by the Bradford method [32].
Results and discussion Fabrication of the CICA system Goat anti-rabbit IgG and PV were immobilized on the inner wall of the glass capillary as control zone and test zone, respectively (see Electronic Supplementary Material (ESM),
a
1.8 13 nm AuNPs-Ab 25 nm AuNPs-Ab 42 nm AuNPs-Ab
1.5 1.2
ABS
optimization of the color development time, the GPTMS modification time was 18 h, and the concentrations of PV and goat anti-rabbit IgG for fabrication at the test zone and control zone were 0.2 mg mL −1 and 0.3 mg mL −1 , respectively.
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0.9 0.6 0.3 0.0
10
100
1000
10000
100000
Dilution ratio
b 0.90
CICA procedures 0.85 0.80
ABS
0.75 0.70 0.65 0.60 0.55 0.50 0
5
10
15
20
25
30
35
40
45
K2CO3 (µL/mL)
c
0.9 0.8 0.7 0.6
ABS
A series of concentrations of PV were prepared in phosphatebuffered saline (PBS, pH 7.4, 0.01 mol L−1), and mixed with the AuNP-labeled anti-PV conjugate at a ratio of 1:1. Then 5 μL of the mixture were taken for use in CICA, as described in the General principle section. For qualitative analysis, the visual detection limit (VDL) was defined as the lowest PV concentration sufficient to form a red band on the test zone significantly weaker than that of controls [33, 34]. For semiquantitative analysis, the CICA result was recorded with a flatbed scanner and the brightness of the signal (negative correlation with actual color) at the test zone (Btest), control zone (Bcontrol), and background (Bbackground, Fig. 1) were calculated by gray-scale image. The relative brightness of the test zone (Brtest) and control zone (Brcontrol) were calculated by subtracting background from the test-zone or control-zone signal, yielding a relative brightness of test zone (Brtest =Btest −Bbackground) and control zone (Brcontrol =Bcontrol −Bbackground), respectively. The calibration curve was fabricated by plotting the relative brightness of the test zone against the logarithm of PV concentration, and the data were fitted to a four-parameter logistic function. The blank was treated as described above, except that PBS was used instead of PV solutions. The semiquantitative limit of detection (LOD) for PV was defined as three times the standard deviation (SD) of the blanks plus the average of the blanks (n=3). For analysis of PV in fish samples, the procedure was the same as that for standard PV, and the quantitative results were calculated from the calibration curve.
0.5 0.4 0.3 0.2 0.1 0
5
10
15
20
25
30
35
Ab (µg/mL) Fig. 2 The effect of reaction conditions on the AuNP-labeled antibody: (a) the effect of the sizes of AuNPs on ELISA, (b) the effect of the AuNP pH on the ABS value of AuNP-labeled antibody, and (c) the effect of the antibody concentrations on the ABS value of AuNP-labeled antibody
Nano-gold capillary immunochromatographic assay for parvalbumin
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binding activity as determined by indirect-ELISA (Fig. 2a), and therefore it was chosen for further experiments. The most popular interpretation of the labeling of antibody with AuNPs is that in alkaline conditions the surface of AuNPs carries negative charge, which can firmly combine with the positively charged groups of antibody by electrostatic adsorption. There is no apparent effect on the biological activity of labeled antibody (see ESM, Fig. S2) [38]. The pH value and the ratio of reagents were believed to be important for the stability of AuNP-labeled antibodies. Here, the absorbance value (ABS) of AuNP-labeled anti-PV antibody increased with the growth of pH until it reached a peak at approximately pH 8.2 (equating to 24 μL 0.1 mol L−1 K2CO3 per milliliter), and then a gradual decrease was observed, indicating increased aggregation of the particles (Fig. 2b). The ABS value was also significantly increased with a higher concentration of antibodies within the range 0 μg mL−1 to 16 μg mL−1, but was very steady when the antibody concentration was more than 20 μg mL−1 (Fig. 2c), which may be caused by equilibration between antibody and AuNPs. Therefore the antibody concentration was optimized at 20 μg mL−1 to react with 1 mL AuNPs. The successful preparation of AuNP-labeled anti-PV antibody was confirmed by UV spectra and TEM analysis. The absorbance peak at 520 nm (Fig. 3a) is usually regarded as characteristic of AuNPs with a diameter of 13 nm [39]. After
Fig. S1). The sample solution containing AuNP-labeled antiPV flowed laterally through the capillary. In the absence of the target, the free antibodies will accumulate at the test zone to form a red band by binding with the immobilized antigen. The excess antibodies will continue flowing until they are captured by the secondary antibodies immobilized at the control zone to form the other red band, which indicates a negative result. However, if there are enough targets in the sample, they will compete with the immobilized antigens to react with the labeled antibodies. Therefore the color at the test zone will be significantly lower than that of controls, and the result will be positive [23]. Because of the directly proportional correlation between the brightness at the test zone and the amount of target in samples within a specific range of concentration, a quantitative analysis can also be performed by imaging techniques [33]. Preparation and characterization of AuNPs and AuNP-labeled antibody Different sizes of AuNP (including those with diameters of 13 nm, 16 nm, 25 nm, and 40 nm) have been used in previous reports for labeling of antibody [24, 35–37]. In this study, three kinds of AuNP (with diameters of 13 nm, 25 nm, and 40 nm) were evaluated; the anti-PV antibody labeled with AuNPs of 13 nm had the highest stability and antigen-
a
0.6 AuNPs labed AuNPs
0.5 0.4 ABS
Fig. 3 Results of UV and TEM analysis of AuNPs and AuNPlabeled anti-PV conjugate: (a) UV spectrums of AuNPs and AuNP-labeled anti-PV conjugate, (b) TEM image of AuNPs, and (c) TEM image of AuNP-labeled anti-PV conjugate
0.3 0.2 0.1 0.0 400
450
500
b
550 λ (nm)
c
600
650
700
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the AuNP–antibody conjugation, an obvious red shift of the absorbance peak from 520 nm to 528 nm was observed (Fig. 3a), and therefore effective conjugation could be confirmed [40]. These results agreed well with the TEM analysis (Figs. 3b, c). Significant shadows were observed around AuNPs (approximately 13 nm) after labeling with antibodies, which clearly indicated the successful preparation of AuNPlabeled anti-PV conjugate. This phenomenon has been observed by other authors studying AuNP-labeled protein conjugate [41–43].
Optimization of the CICA performance When the GPTMS modification time of the capillary was in the range 4–18 h, the calculated relative brightness decreased sharply; it became steady when the modification time was in the range 18–48 h (ESM, Fig. S3a). In principle such a result should be attributed to the increase of epoxy groups on the inner wall of the glass capillary, which will result in increased immobilization of PV to capture more AuNP-labeled antibodies on the test zone. But it was clearly revealed that epoxy groups could be essentially saturated after 18 h of modification; an optimized modification time of 18 h was therefore chosen. The highest sensitivity and minimum immunoreagent consumption are always taken as the criteria for optimization of immunoassays. For the CICA, the concentrations of goat anti-rabbit IgG and PV should make the color of the control zone the same as that of the test zone for blanks and make the control zone completely colorless for positive samples with a target concentration as low as possible. On the basis of these criteria, a series of concentrations of goat anti-rabbit IgG and PV were immobilized on the control zone and test zone, respectively, which resulted in remarkable differences for blank detection. On increasing PV coating concentration from 0.1–0.3 mg mL−1 (see ESM, Table S1), the red color changed from invisible to crimson. The color on the test zone was clearly visible with a PV-coating concentration of 0.2 mg mL−1, and was the same as the color of the test zone with a goat anti-rabbit IgG coating concentration of 0.3 mg mL−1. At the same concentrations, a complete lack of color was observed on the test zone for positive samples. Therefore, the concentrations of goat anti-rabbit IgG and of PV were optimized at 0.3 mg mL−1 and 0.2 mg mL−1, respectively. The color development time was also revealed to be important for the CICA performance. For negative samples, the relative brightness reduced rapidly when the incubation time increased from 1 min to 4 min, and then leveled off slowly as the time increased further (see ESM, Fig. S3b), indicating that 4 min was sufficient for color stability .
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Efficiency evaluation of the CICA The efficiency of the developed CICA was preliminarily evaluated in buffers, using PV as an analyte. For qualitative analysis, the VDL for PV was estimated to be approximately 70 ng mL−1 (Fig. 4a). The CICA result can also be quantitatively or semi-quantitatively analyzed by determination of the brightness at the test zone and background. On the basis of the calibration curve (Fig. 4b), the semi-quantitative LOD was calculated to be 40 ng mL−1. Subsequently, the linearity in the range 50–1000 ng mL−1 was obtained between the relative brightness and different concentrations of PV (see ESM, Fig. S4). On the basis of the currently recommended threshold value of 5 mg kg−1 for allergic consumers [44], both the qualitative and quantitative sensitivity of the CICA seem sufficient for effective screening of PV in foodstuffs. The reproducibility and repeatability of the developed CICA were evaluated by three duplicated quantitative measurements of seven batches with the same PV concentration (50 ng mL−1). The intra-assay coefficient of variation (CV) ranged from 2.7 to 9.7 %, and the interassay CV was 8.0 %. The stability of the assay was examined by using the same batch of CICA for analysis of PV at a concentration of 50 ng mL−1. After four weeks of storage at 4 °C, the brightness of the test zone retained 94.6 % of its original level. This stability was much better than that of immunochromatographic assays performed on NCM, and could therefore greatly facilitate the preservation. The reasons may be the excellent stability of the glass capillary and its effective protection of the test zone from environmental conditions including oxygen, humidity, and light. The covalent binding of the PV on the glass via epoxy groups, which is believed to be firmer than the usually exploited electronic attractions and hydrophobic interactions [45, 46], may also improve the stability of CICA. To test the specificity of the assay, human serum albumin and egg albumin were detected as negative controls. Meanwhile, extracts from several aquatic foods and from other foods including beef, pork, and chicken that were supposed to contain PV were tested. Standard solutions of negative control at concentrations of 1, 10, 100, and 1000 μg mL−1 were detected by the CICA. Compared with the blank control, no significant color change of the test zone was observed (see ESM, Fig. S5). It was not surprising to observe that antiparvalbumin antibody could cross-react with PV from surimi products, as shown in Table 1. These surimi products were also positively detected by the CICA (see ESM, Fig. S6a). No cross-reactivity was observed with samples from beef, pork, and chicken (see ESM, Fig. S6b), strongly indicating the high specificity of the developed CICA.
Nano-gold capillary immunochromatographic assay for parvalbumin Fig. 4 CICA results for parvalbumin in PBS: (a) test zones with PV concentrations from 0 to 106 ng mL−1, from left to right, and (b) calibration curve obtained by fitting a fourparameter logistic function, where each point is the average of a triplicate
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a Control zone
Test zone 0
10
30
50
70
90 100 200 300
103 104 105
400 500
106
ng/mL
b 0
Relative brightness
-6
-12
-18
-24
Model
Boltzmann
Equation
y = A2 + (A1-A 2)/(1 + exp((x-x 0)/dx))
Reduced Chi-Sqr
1.59192
Adj. R-Square
0.99396 Value
B B B B
-30
0
101
102
A1 A2 x0 dx
Standard Error
-31.42513 -1.73111 2.24106 0.27947
104 103 C(ng/mL)
105
0.7823 0.34509 0.03834 0.02636
106
Table 1 Determination of parvalbumin (PV) in different food samples with 100–105-fold dilution, using the CICA method Dilution ratio
Scophthalmus maximus (2.19 mg mL−1 )
Fish ball I
Cuttlefish ball
Fish ball II
Fish tofu
Beef
Pork
Chicken
100
+++a
+++
+++
+++
+++
−−−
−−−
−−−
1
+++ +++ +++ −−−b −−−
+++ +++ −−− −−− –
+++ ±c + ± −−− −−− –
+++ +++ −−− −−− –
+++ +++ −−− −−− –
−−− – – – –
−−− – – – –
−−− – – – –
10 102 103 104 105 a
“positive”, PV concentration more than 70 ng mL−1
b
“negative”, PV concentration below 70 ng mL−1
c
“positive/negative”, PV concentration approximately 70 ng mL−1
6644 Table 2 Recovery of CICA for beef, pork, and chicken spiked with parvalbumin (PV)
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Sample
Spiked PV (ng mL−1)
Calculated PV (ng mL−1)
CV (%, n=6)
Recovery (%)
Beef
0
ND
–
–
100
112
5
112
200 0
223 ND
9 –
111 –
100
109
7
109
200 0
253 ND
10 –
127 –
100
110
13
110
200
250
–
125
Pork
Chicken
Validation with real samples The efficiency of CICA for analysis of real samples was validated with different foodstuffs. PV has been proved to be of high thermostability [47]. Here, considering the high purity of the PV in Scophthalmus maximus (Linnaeus) samples after heating at 98 °C for 5 min (see ESM, Fig. S7a), its concentration was approximately estimated as the total protein concentration, which was calculated to be approximately 2.19 mg mL−1. With serial 10-fold dilution of the sample, PV was effectively detected at a concentration of 219 ng mL−1 after more than 104-fold dilution (Table 1), which agreed well with the VDL of the technique. This result also indicated a satisfactory resistance of the CICA to matrix interference, and this will greatly increase its reliability for trace targets. The presence of PV was also detected by CICA in other four fish products (including fish balls and fish tofu), even after 10–100-fold dilution of the samples, but for pork, beef, and chicken (with no fish component listed on the label) the result was negative (Table 1). Although the efficiency of CICA for quantitative determination seemed difficult to evaluate because of the uncertain PV concentration in the complex matrix, it could indicate that the ingredients of surimi might contain traces of PV. SDS-PAGE analysis (see ESM, Fig. S7b)
Table 3 Efficiency of different immunoassays for parvalbumin
agreed well with the CICA results for PV in fish products (see ESM, Fig. S6a), and revealed that small amounts of PV existed in the extracts of fish products. However, there was no PV band in the SDS-PAGE analysis (see ESM, Fig. S7c) of the extracts from pork, beef, and chicken, for which the CICA also obtained negative results (see ESM, Fig. S6b). Therefore, it was confirmed that the CICA can be used as a qualitative screening method for PV in complex food samples. The accuracy of the developed CICA was evaluated using beef, pork, and chicken spiked with different concentrations of PV. As shown in Table 2, no PV was detected in blank samples, and the recovery ranged from 108.96 to 126.53 % at the PV-spiking concentrations of 100 ng mL−1 and 200 ng mL−1. This result was very close to the usually recognized acceptable level (80–120 %) [4] for such techniques, but a little higher. This may be attributed to many possible causes; for example, there may be cross-reactivity between the antiparvalbumin antibody and a trace substance that was not present in sufficient quantities to be detected in unspiked samples. Such cross-reactivity can result in a significant increase in the recovery, similar to results reported in [4]. Moreover, as we observed for some fish samples, a component in the beef and pork may interfere with the accuracy of the immunoassay; this is usually named the “matrix effect” [31].
Methods
Detection time
Linear range (μg L−1)
Detection limit (μg L−1)
Ref.
Lateral-flow immunoassay
20 min
10–105
LOD 500
[4]
Sandwich ELISA
1–2 h
0.49–500
LOQ 46 LOD 0.11
[2]
0.625–10,240
LOQ 0.22 LOD 0.8
[48] [49] [50] This work
Competitive ELISA
1–2 h
Sandwich ELISA
1–2 h
0.78–50
LOQ 5.59 LOD 0.58
SPR CICA
5 min 30 min
– 50–1000
LOQ 1.76 3.55 LOD 70 LOQ 40
Nano-gold capillary immunochromatographic assay for parvalbumin
There may be other, unknown reasons for higher recovery in samples, warranting further discussion. With modification of anti-PV antibodies and optimization of sample pretreatment in the next work, one may expect significantly improved accuracy of the CICA. The efficiency of CICA and other immunoassays used for the detection of PV is summarized in Table 3. Considering the difference in antibodies, PV, food samples, and the definition of the detection limit among these techniques, it seems very difficult to make an accurate comparison of the sensitivity. Although the LOD of CICA did not reach the sensitivity of ELISA, the whole detection time was greatly shortened because of the simple pretreatment process. Also, the non-instrumental bioanalysis of CICA reduced the cost and reduced the technical requirements for testing personnel. Furthermore, the rapidity of CICA was similar to that of a quantitative lateral-flow immunoassay based on superparamagnetic nanoparticles for parvalbumin, but the LOD of CICA was obviously lower than that of the lateral-flow immunoassay. Although the LOQ of CICA was approximately that of the lateral-flow immunoassay, the simple and fast quantitative-detection instrument, excellent dynamic range, and good stability enable us to suggest the CICA to be an effective tool for routine monitoring of trace PV in foodstuffs. Concerning the performance time, both immunochromatographic assays were much faster than traditional ELISA techniques, indicating their promising potential for screening large numbers of samples. The fabrication cost was estimated to be ∼$0.53, with the most expensive material being anti-parvalbumin antibody (see ESM, Table S2) to enable the rapid detection of PV by the naked eye. The equipment costs, including a flatbed scanner and computer, were not included in this calculation. A more accurate estimate of the fabrication cost for semi-quantitative analysis should be calculated on the basis of the in-situ test conditions.
Conclusion Using glass capillary as the carrier, a new nano-gold immunochromatographic assay (CICA) was designed and fabricated. Using PV as the analyte, the system was optimized and its efficiency was fully evaluated and validated. In comparison to traditional immunoassays, including ELISA and NCM-based immunochromatographic techniques, the developed CICA had significant advantages, including improved sensitivity, simplicity, speed, and stability. All these results enabled us to suggest the CICA to have good potential as a new and effective technique for fast screening of biological samples.
6645 Acknowledgement This work was supported by the “Earmarked Fund for China Agriculture Research System (CARS-50)” and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1188).
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RESEARCH PAPER
Nano-gold capillary immunochromatographic assay for parvalbumin Shuyuan Du & Hong Lin & Jianxin Sui & Xiudan Wang & Limin Cao
Received: 17 June 2014 / Revised: 2 August 2014 / Accepted: 5 August 2014 / Published online: 29 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract A novel non-instrumental bioanalysis based on colloidal-gold immunochromatography in a modified glass capillary was developed and named capillary immunochromatographic assay (CICA). In this report, glass capillary was proposed as a support in immunochromatographic assay because of its excellent characteristics. Goat anti-rabbit IgG and parvalbumin (PV) were immobilized on the inner wall of the glass capillary as control zone and test zone, respectively. The CICA was constructed, and main variables for the performance were optimized. Using an important allergen of fish products (parvalbumin, PV) as the target, the analytical efficiency of the developed technique was investigated and the visual detection limit (VDL) and semi-quantitative limit of detection (LOD) were estimated to be 70 ng mL−1 and 40 ng mL−1, respectively. The coefficient of variation (CV) for the intra-assay and inter-assay was calculated for the PV concentration of 50 ng mL−1, and the entire operation, including sample preparation, was consistently performed in 30 min. The developed technique was implemented and validated with different foodstuffs, including Scophthalmus maximus (Linnaeus), surimi products, and livestock, confirming sufficient accuracy and precision of results and verifying the method to be efficacious. These results enabled us to propose CICA as a new and promising technique for simple, rapid, and on-site screening of PV in biological samples.
Electronic supplementary material The online version of this article (doi:10.1007/s00216-014-8093-0) contains supplementary material, which is available to authorized users. S. Du : H. Lin : J. Sui : X. Wang : L. Cao (*) Food Safety Laboratory, Ocean University of China, Qingdao 266003, China e-mail: [email protected]
Keywords Capillary . Capillary immunochromatographic assay . Colloidal gold . Parvalbumin
Introduction Recently a variety of immunoassays have been developed in the fields of food, environmental analysis, and clinical applications. Traditional techniques, including immunosensors and enzyme-linked immunosorbent assay (ELISA), usually have satisfactory sensitivity, accuracy, selectivity, and precision [1–3]. However, they are relatively complex, time consuming, and largely dependent on professional equipment and operating experience [4–7], meaning their cost and complexity limits their practical applications, especially in field detection. Hence, there is increasing attention focusing on investigating techniques, for example immunochromatographic assays, that can improve the simplicity by using new low-cost and high-performance materials. In comparison to ELISA and other immunoassays, such techniques have unique advantages [8–10]: the operation (after sample pretreatment) can be accomplished in less than 20 min, and the final results can be qualitatively detected by the naked eye without any need of equipment. Most of these techniques, for example lateral-flow colloidal-gold immunoassay (LFIA) and dot-immunogold filtration assay (DIGFA), were developed using nitrocellulose membrane (NCM) as the carrier material. The high proteinbinding capacity of NCM may facilitate the immobilization of antibodies. However, NCM is subject to breakage, compression, and scoring during processing, and is also greatly affected by environmental conditions including temperature and relative humidity [11]. Towing phenomena, diffusion phenomena, and significant deviation (inter and intra) always exist in chromatography because of the complicated and heterogeneous structure of NCM. For these reasons, NCM-based immunochromatographic assays have relatively poor
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sensitivity, reproducibility, and repeatability, which limits their use in real applications. Other materials have recently been used in immunochromatographic assays as alternatives to NCM, including paper [12–15], thread [16, 17], and an SPE column [18]. Although these new techniques still seem far from commercial application, they provide very valuable ideas regarding development of more reliable, convenient, and efficient immunochromatographic techniques. In this study, a new strategy is proposed for the design and fabrication of an immunochromatographic assay based on a glass capillary. This is mainly motivated by the unique advantages of the glass capillary in comparison to other materials: low cost, less sample usage, easily modified, and favorable optical properties. More importantly, the uniform smooth surface and the high rigidity and ionic strength tolerance of the glass matrix make it more robust than flexible materials regarding possible interference from the environment and the sample matrix, which can be very helpful for improving the accuracy and precision of the analytical operations. Herein, a nano-gold capillary immunochromatographic assay (CICA) emulating the lateral-flow format was designed and fabricated, the main variables affecting the CICA response were investigated, and the performing conditions were optimized. Using an allergen (parvalbumin) in fish products as the target, the real efficiency of the technique for analyzing biological samples was validated, and its potential use as a new screening tool was also evaluated and discussed.
Materials and methods Reagents Parvalbumin and anti-parvalbumin antibody with an affinity constant of 1.58×109 L mol−1 were prepared and characterized as in our previous work [19–22]. Goat anti-rabbit IgG was purchased from Zhongshan Jinqiao Co. (Beijing, China). 3-Glycidyloxypropyltrimethoxysilane (98 %, GPTMS) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Hydrogen tetrachloroaurate (III) (HAuCl4), trisodium citrate, toluene, acetone, and triethylamine (TEA) were obtained from National Chemical Pharmaceutical Co. (Shanghai, China). Bovine serum albumin (BSA), human serum albumin, egg albumin, tris(hydroxymethylamino) metane (Tris), and Tween 20 were purchased from Solarbio (Beijing, China). Molecularweight markers were purchased from Fermantas Co. Ltd (Vilnius, Lithuania). All chemicals were of analytical grade unless otherwise stated. Apparatus The glass capillary (d=0.9 mm) was purchased from the instrument factory of West China University of Medical
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Sciences (Chengdu, China). The protein A column was purchased from GE Healthcare (GE, USA). The UV–vis spectra were recorded with a UV-1101 from Techcomp (Shanghai, China). The AuNPs and AuNP-labeled anti-PV conjugates were characterized by transmission electron microscope (TEM, JEOL JEM-1200EX, Japan). All centrifugations were performed with Z36HK (HERMLE Labortechnik GmbH, Germany). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results were recorded on a Tanon180 Gel imaging system (Tanon Science & Technology Co. Ltd. Shanghai, P. R. China). After immunochromatographic reaction, the images were scanned by an HP Scanjet G4050 scanner from Seiko Epson Nagano (HP, Japan) to obtain the brightness of the control and test zones. All aqueous solutions were prepared in ultra-pure water of 18.2 MΩ purified by Unique-R20 from Research Scientific Instruments Co. (Beijing, China). General principle The CICA system was constructed using the principle of direct competitive immunoassay. The PV and goat antirabbit IgG were immobilized on the inner wall of the glass capillary by covalent bonding to form the test zone (one end of capillary) and control zone (other end of capillary), respectively (Fig. 1a). The capillary was placed horizontally on a table, and a mixture of AuNP-labeled anti-PV and analyte was injected by a pipettor into the modified capillary to fully cover the test zone, and reacted for 4 min. Then the solution was forced to the control zone by the pipettor, and reacted for another 4 min. The unbound conjugate was removed from the capillary by pipettor, and the capillary was washed thoroughly by moving up and down in phosphate-buffered saline with Tween 20 (PBST, 1 g L−1 Tween 20 in 0.01 mol L−1 PBS, pH 7.4). For negative samples, two red bands appeared as a result of the accumulation of red AuNP-labeled anti-PV conjugate at both zones, and their brightness was almost the same (Fig. 1b) [23]. In contrast, for the positive samples the brightness of the test zone was visually weaker than that of the control zone, sometimes to the extent that no significant red band appeared (Fig. 1c). Preparation of AuNPs and AuNP-labeled anti-parvalbumin antibody AuNPs were prepared according to the literature, with slight modifications [24]. The size of AuNPs was controlled by subtle variation of the ratio of HAuCl4 to sodium citrate in the mixture. When the color of the solution stabilized, boiling continued for another 15 min. The mixture was heated under reflux to room temperature, and then stored at 4 °C in a dark bottle until further use. The AuNP-labeled anti-PV conjugate was prepared according to [25]. The crude anti-PV was purified by a protein
Nano-gold capillary immunochromatographic assay for parvalbumin Fig. 1 The scheme of the CICA system: (a) the control zone and test zone on the capillary, (b) negative results and (c) positive results
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A column according to the instructions, and then centrifuged at 3000g to remove the precipitation. The optimum pH and concentration ratio of antibody to AuNP were determined by the Slot method [26]. The AuNPs were adjusted to the optimum pH with K2CO3 (0.1 mol L−1), and the anti-PV antibody was slowly added to the AuNPs with continuous stirring. The mixture was stirred gently for 2 h, and subsequently 10 % BSA and 1 % polyethylene glycol (PEG)-20000 were added to reach the final concentrations of 1.0 % (w/v) and 10 % (v/v), respectively. Then the mixture was reacted at room temperature for 30 min to block the residual surface of AuNPs, followed by centrifugation at 2500g for 15 min at 4 °C. The supernatant was further centrifuged at 9000–11,000g for 1 h at 4 °C. Finally, the precipitation was suspended in Tris–HCl (0.01 mol L−1, pH 8.2, containing 1 % BSA and 0.02 % NaN3) and stored at 4 °C until further use. The formation of AuNPs and AuNP-labeled anti-PV conjugate was checked with UV– vis spectra and TEM in Medical College of Qingdao University. For characterization of AuNP-labeled anti-PV conjugate, an indirect ELISA was performed as described in [27]. The variables were first optimized by checkerboard titrations [28], and the concentrations of PV and the HRP-labeled antibody were optimized as 10 μg mL−1 and 1.6 μg mL−1, respectively. Pretreatment and modification of the capillary The pretreatment and modification of the capillary were performed by the procedure described in [29], with modification. The glass capillary was immersed in a mixture of “piranha solution” (H2SO4:30 % H2O2 =3:1, below 80 °C) and sonicated for 15 min. Then it was rinsed thoroughly with deionized water until the pH of the cleansing solution was close to 7.0, and dried at 105 °C for approximately 60 min. The capillary was
goat anti-rabbit IgG
Positive test
then successively washed with KOH (1 mol L−1), water, HCl (1 mol L−1), water, and acetone, each for 15 min. Finally the capillary was dried at 105 °C for more than 1 h. After cleaning the capillary was modified immediately by the following procedure. It was first immersed in GPTMS solution (10 %, v/v in dry acetone, containing 1 % triethylamine) for 18 h at room temperature. Then the GPTMS solution in the beaker was removed and the reaction was continued for another 2 h. The capillary was washed thoroughly by moving up and down in toluene and acetone for 5 min, and dried by flushing with nitrogen. Then 3 μL PV was injected into one end of the capillary by pipettor to fabricate the test zone. The capillary was placed vertically in a well of ELISA plate at 25 °C for 2 h to immobilize antigen and fabricate the test zone on the inner wall. Redundant PV was removed from the capillary by pipettor, and the modified capillary was washed by immersing in PBST and moving up and down for 5 min. 3 μL goat anti-rabbit IgG was injected into the control-zone end of the capillary by pipettor. The control zone was fabricated on the inside wall of the capillary using the same procedure as for the test zone. After removing redundant goat anti-rabbit IgG and washing with PBST, the modified capillary was immersed in 1.5 % BSA for 2 h at 37 °C to block unspecific sites. Finally, the capillary was rinsed by PBST again as described above and stored at 4 °C until use. For optimization of the GPTMS modification time (in the range 4–48 h), the concentrations of PV and goat anti-rabbit IgG were 0.4 mg mL−1, and the color development time was 8 min. For optimization of the concentration of PV and goat anti-rabbit IgG for immobilization on the capillary, the GPTMS modification time was 18 h, the color development time was 8 min, and PBS was used for the blank. For
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Sample preparation Scophthalmus maximus (Linnaeus), surimi products, and livestock and poultry meat samples were collected from a local supermarket (Qingdao, China). The pretreatment of samples used a method described elsewhere, with modifications [30]. First, the samples were homogenized with Tris–HCl (10 mmol L−1, pH 7.5) at a ratio of 1:2 (w/v), followed by heating in a water bath at 98 °C for 5 min. Then the mixture was centrifuged at 3800g for 5 min and the supernatant was collected. The protein content of the extract was investigated by discontinuous SDS-PAGE under denatured conditions using a procedure described in [31], and the results were recorded using an HP Scanjet G4050 (Hewlett-Packard). The protein content of the prepared samples was determined by the Bradford method [32].
Results and discussion Fabrication of the CICA system Goat anti-rabbit IgG and PV were immobilized on the inner wall of the glass capillary as control zone and test zone, respectively (see Electronic Supplementary Material (ESM),
a
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optimization of the color development time, the GPTMS modification time was 18 h, and the concentrations of PV and goat anti-rabbit IgG for fabrication at the test zone and control zone were 0.2 mg mL −1 and 0.3 mg mL −1 , respectively.
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A series of concentrations of PV were prepared in phosphatebuffered saline (PBS, pH 7.4, 0.01 mol L−1), and mixed with the AuNP-labeled anti-PV conjugate at a ratio of 1:1. Then 5 μL of the mixture were taken for use in CICA, as described in the General principle section. For qualitative analysis, the visual detection limit (VDL) was defined as the lowest PV concentration sufficient to form a red band on the test zone significantly weaker than that of controls [33, 34]. For semiquantitative analysis, the CICA result was recorded with a flatbed scanner and the brightness of the signal (negative correlation with actual color) at the test zone (Btest), control zone (Bcontrol), and background (Bbackground, Fig. 1) were calculated by gray-scale image. The relative brightness of the test zone (Brtest) and control zone (Brcontrol) were calculated by subtracting background from the test-zone or control-zone signal, yielding a relative brightness of test zone (Brtest =Btest −Bbackground) and control zone (Brcontrol =Bcontrol −Bbackground), respectively. The calibration curve was fabricated by plotting the relative brightness of the test zone against the logarithm of PV concentration, and the data were fitted to a four-parameter logistic function. The blank was treated as described above, except that PBS was used instead of PV solutions. The semiquantitative limit of detection (LOD) for PV was defined as three times the standard deviation (SD) of the blanks plus the average of the blanks (n=3). For analysis of PV in fish samples, the procedure was the same as that for standard PV, and the quantitative results were calculated from the calibration curve.
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Ab (µg/mL) Fig. 2 The effect of reaction conditions on the AuNP-labeled antibody: (a) the effect of the sizes of AuNPs on ELISA, (b) the effect of the AuNP pH on the ABS value of AuNP-labeled antibody, and (c) the effect of the antibody concentrations on the ABS value of AuNP-labeled antibody
Nano-gold capillary immunochromatographic assay for parvalbumin
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binding activity as determined by indirect-ELISA (Fig. 2a), and therefore it was chosen for further experiments. The most popular interpretation of the labeling of antibody with AuNPs is that in alkaline conditions the surface of AuNPs carries negative charge, which can firmly combine with the positively charged groups of antibody by electrostatic adsorption. There is no apparent effect on the biological activity of labeled antibody (see ESM, Fig. S2) [38]. The pH value and the ratio of reagents were believed to be important for the stability of AuNP-labeled antibodies. Here, the absorbance value (ABS) of AuNP-labeled anti-PV antibody increased with the growth of pH until it reached a peak at approximately pH 8.2 (equating to 24 μL 0.1 mol L−1 K2CO3 per milliliter), and then a gradual decrease was observed, indicating increased aggregation of the particles (Fig. 2b). The ABS value was also significantly increased with a higher concentration of antibodies within the range 0 μg mL−1 to 16 μg mL−1, but was very steady when the antibody concentration was more than 20 μg mL−1 (Fig. 2c), which may be caused by equilibration between antibody and AuNPs. Therefore the antibody concentration was optimized at 20 μg mL−1 to react with 1 mL AuNPs. The successful preparation of AuNP-labeled anti-PV antibody was confirmed by UV spectra and TEM analysis. The absorbance peak at 520 nm (Fig. 3a) is usually regarded as characteristic of AuNPs with a diameter of 13 nm [39]. After
Fig. S1). The sample solution containing AuNP-labeled antiPV flowed laterally through the capillary. In the absence of the target, the free antibodies will accumulate at the test zone to form a red band by binding with the immobilized antigen. The excess antibodies will continue flowing until they are captured by the secondary antibodies immobilized at the control zone to form the other red band, which indicates a negative result. However, if there are enough targets in the sample, they will compete with the immobilized antigens to react with the labeled antibodies. Therefore the color at the test zone will be significantly lower than that of controls, and the result will be positive [23]. Because of the directly proportional correlation between the brightness at the test zone and the amount of target in samples within a specific range of concentration, a quantitative analysis can also be performed by imaging techniques [33]. Preparation and characterization of AuNPs and AuNP-labeled antibody Different sizes of AuNP (including those with diameters of 13 nm, 16 nm, 25 nm, and 40 nm) have been used in previous reports for labeling of antibody [24, 35–37]. In this study, three kinds of AuNP (with diameters of 13 nm, 25 nm, and 40 nm) were evaluated; the anti-PV antibody labeled with AuNPs of 13 nm had the highest stability and antigen-
a
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Fig. 3 Results of UV and TEM analysis of AuNPs and AuNPlabeled anti-PV conjugate: (a) UV spectrums of AuNPs and AuNP-labeled anti-PV conjugate, (b) TEM image of AuNPs, and (c) TEM image of AuNP-labeled anti-PV conjugate
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the AuNP–antibody conjugation, an obvious red shift of the absorbance peak from 520 nm to 528 nm was observed (Fig. 3a), and therefore effective conjugation could be confirmed [40]. These results agreed well with the TEM analysis (Figs. 3b, c). Significant shadows were observed around AuNPs (approximately 13 nm) after labeling with antibodies, which clearly indicated the successful preparation of AuNPlabeled anti-PV conjugate. This phenomenon has been observed by other authors studying AuNP-labeled protein conjugate [41–43].
Optimization of the CICA performance When the GPTMS modification time of the capillary was in the range 4–18 h, the calculated relative brightness decreased sharply; it became steady when the modification time was in the range 18–48 h (ESM, Fig. S3a). In principle such a result should be attributed to the increase of epoxy groups on the inner wall of the glass capillary, which will result in increased immobilization of PV to capture more AuNP-labeled antibodies on the test zone. But it was clearly revealed that epoxy groups could be essentially saturated after 18 h of modification; an optimized modification time of 18 h was therefore chosen. The highest sensitivity and minimum immunoreagent consumption are always taken as the criteria for optimization of immunoassays. For the CICA, the concentrations of goat anti-rabbit IgG and PV should make the color of the control zone the same as that of the test zone for blanks and make the control zone completely colorless for positive samples with a target concentration as low as possible. On the basis of these criteria, a series of concentrations of goat anti-rabbit IgG and PV were immobilized on the control zone and test zone, respectively, which resulted in remarkable differences for blank detection. On increasing PV coating concentration from 0.1–0.3 mg mL−1 (see ESM, Table S1), the red color changed from invisible to crimson. The color on the test zone was clearly visible with a PV-coating concentration of 0.2 mg mL−1, and was the same as the color of the test zone with a goat anti-rabbit IgG coating concentration of 0.3 mg mL−1. At the same concentrations, a complete lack of color was observed on the test zone for positive samples. Therefore, the concentrations of goat anti-rabbit IgG and of PV were optimized at 0.3 mg mL−1 and 0.2 mg mL−1, respectively. The color development time was also revealed to be important for the CICA performance. For negative samples, the relative brightness reduced rapidly when the incubation time increased from 1 min to 4 min, and then leveled off slowly as the time increased further (see ESM, Fig. S3b), indicating that 4 min was sufficient for color stability .
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Efficiency evaluation of the CICA The efficiency of the developed CICA was preliminarily evaluated in buffers, using PV as an analyte. For qualitative analysis, the VDL for PV was estimated to be approximately 70 ng mL−1 (Fig. 4a). The CICA result can also be quantitatively or semi-quantitatively analyzed by determination of the brightness at the test zone and background. On the basis of the calibration curve (Fig. 4b), the semi-quantitative LOD was calculated to be 40 ng mL−1. Subsequently, the linearity in the range 50–1000 ng mL−1 was obtained between the relative brightness and different concentrations of PV (see ESM, Fig. S4). On the basis of the currently recommended threshold value of 5 mg kg−1 for allergic consumers [44], both the qualitative and quantitative sensitivity of the CICA seem sufficient for effective screening of PV in foodstuffs. The reproducibility and repeatability of the developed CICA were evaluated by three duplicated quantitative measurements of seven batches with the same PV concentration (50 ng mL−1). The intra-assay coefficient of variation (CV) ranged from 2.7 to 9.7 %, and the interassay CV was 8.0 %. The stability of the assay was examined by using the same batch of CICA for analysis of PV at a concentration of 50 ng mL−1. After four weeks of storage at 4 °C, the brightness of the test zone retained 94.6 % of its original level. This stability was much better than that of immunochromatographic assays performed on NCM, and could therefore greatly facilitate the preservation. The reasons may be the excellent stability of the glass capillary and its effective protection of the test zone from environmental conditions including oxygen, humidity, and light. The covalent binding of the PV on the glass via epoxy groups, which is believed to be firmer than the usually exploited electronic attractions and hydrophobic interactions [45, 46], may also improve the stability of CICA. To test the specificity of the assay, human serum albumin and egg albumin were detected as negative controls. Meanwhile, extracts from several aquatic foods and from other foods including beef, pork, and chicken that were supposed to contain PV were tested. Standard solutions of negative control at concentrations of 1, 10, 100, and 1000 μg mL−1 were detected by the CICA. Compared with the blank control, no significant color change of the test zone was observed (see ESM, Fig. S5). It was not surprising to observe that antiparvalbumin antibody could cross-react with PV from surimi products, as shown in Table 1. These surimi products were also positively detected by the CICA (see ESM, Fig. S6a). No cross-reactivity was observed with samples from beef, pork, and chicken (see ESM, Fig. S6b), strongly indicating the high specificity of the developed CICA.
Nano-gold capillary immunochromatographic assay for parvalbumin Fig. 4 CICA results for parvalbumin in PBS: (a) test zones with PV concentrations from 0 to 106 ng mL−1, from left to right, and (b) calibration curve obtained by fitting a fourparameter logistic function, where each point is the average of a triplicate
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Table 1 Determination of parvalbumin (PV) in different food samples with 100–105-fold dilution, using the CICA method Dilution ratio
Scophthalmus maximus (2.19 mg mL−1 )
Fish ball I
Cuttlefish ball
Fish ball II
Fish tofu
Beef
Pork
Chicken
100
+++a
+++
+++
+++
+++
−−−
−−−
−−−
1
+++ +++ +++ −−−b −−−
+++ +++ −−− −−− –
+++ ±c + ± −−− −−− –
+++ +++ −−− −−− –
+++ +++ −−− −−− –
−−− – – – –
−−− – – – –
−−− – – – –
10 102 103 104 105 a
“positive”, PV concentration more than 70 ng mL−1
b
“negative”, PV concentration below 70 ng mL−1
c
“positive/negative”, PV concentration approximately 70 ng mL−1
6644 Table 2 Recovery of CICA for beef, pork, and chicken spiked with parvalbumin (PV)
S. Du et al.
Sample
Spiked PV (ng mL−1)
Calculated PV (ng mL−1)
CV (%, n=6)
Recovery (%)
Beef
0
ND
–
–
100
112
5
112
200 0
223 ND
9 –
111 –
100
109
7
109
200 0
253 ND
10 –
127 –
100
110
13
110
200
250
–
125
Pork
Chicken
Validation with real samples The efficiency of CICA for analysis of real samples was validated with different foodstuffs. PV has been proved to be of high thermostability [47]. Here, considering the high purity of the PV in Scophthalmus maximus (Linnaeus) samples after heating at 98 °C for 5 min (see ESM, Fig. S7a), its concentration was approximately estimated as the total protein concentration, which was calculated to be approximately 2.19 mg mL−1. With serial 10-fold dilution of the sample, PV was effectively detected at a concentration of 219 ng mL−1 after more than 104-fold dilution (Table 1), which agreed well with the VDL of the technique. This result also indicated a satisfactory resistance of the CICA to matrix interference, and this will greatly increase its reliability for trace targets. The presence of PV was also detected by CICA in other four fish products (including fish balls and fish tofu), even after 10–100-fold dilution of the samples, but for pork, beef, and chicken (with no fish component listed on the label) the result was negative (Table 1). Although the efficiency of CICA for quantitative determination seemed difficult to evaluate because of the uncertain PV concentration in the complex matrix, it could indicate that the ingredients of surimi might contain traces of PV. SDS-PAGE analysis (see ESM, Fig. S7b)
Table 3 Efficiency of different immunoassays for parvalbumin
agreed well with the CICA results for PV in fish products (see ESM, Fig. S6a), and revealed that small amounts of PV existed in the extracts of fish products. However, there was no PV band in the SDS-PAGE analysis (see ESM, Fig. S7c) of the extracts from pork, beef, and chicken, for which the CICA also obtained negative results (see ESM, Fig. S6b). Therefore, it was confirmed that the CICA can be used as a qualitative screening method for PV in complex food samples. The accuracy of the developed CICA was evaluated using beef, pork, and chicken spiked with different concentrations of PV. As shown in Table 2, no PV was detected in blank samples, and the recovery ranged from 108.96 to 126.53 % at the PV-spiking concentrations of 100 ng mL−1 and 200 ng mL−1. This result was very close to the usually recognized acceptable level (80–120 %) [4] for such techniques, but a little higher. This may be attributed to many possible causes; for example, there may be cross-reactivity between the antiparvalbumin antibody and a trace substance that was not present in sufficient quantities to be detected in unspiked samples. Such cross-reactivity can result in a significant increase in the recovery, similar to results reported in [4]. Moreover, as we observed for some fish samples, a component in the beef and pork may interfere with the accuracy of the immunoassay; this is usually named the “matrix effect” [31].
Methods
Detection time
Linear range (μg L−1)
Detection limit (μg L−1)
Ref.
Lateral-flow immunoassay
20 min
10–105
LOD 500
[4]
Sandwich ELISA
1–2 h
0.49–500
LOQ 46 LOD 0.11
[2]
0.625–10,240
LOQ 0.22 LOD 0.8
[48] [49] [50] This work
Competitive ELISA
1–2 h
Sandwich ELISA
1–2 h
0.78–50
LOQ 5.59 LOD 0.58
SPR CICA
5 min 30 min
– 50–1000
LOQ 1.76 3.55 LOD 70 LOQ 40
Nano-gold capillary immunochromatographic assay for parvalbumin
There may be other, unknown reasons for higher recovery in samples, warranting further discussion. With modification of anti-PV antibodies and optimization of sample pretreatment in the next work, one may expect significantly improved accuracy of the CICA. The efficiency of CICA and other immunoassays used for the detection of PV is summarized in Table 3. Considering the difference in antibodies, PV, food samples, and the definition of the detection limit among these techniques, it seems very difficult to make an accurate comparison of the sensitivity. Although the LOD of CICA did not reach the sensitivity of ELISA, the whole detection time was greatly shortened because of the simple pretreatment process. Also, the non-instrumental bioanalysis of CICA reduced the cost and reduced the technical requirements for testing personnel. Furthermore, the rapidity of CICA was similar to that of a quantitative lateral-flow immunoassay based on superparamagnetic nanoparticles for parvalbumin, but the LOD of CICA was obviously lower than that of the lateral-flow immunoassay. Although the LOQ of CICA was approximately that of the lateral-flow immunoassay, the simple and fast quantitative-detection instrument, excellent dynamic range, and good stability enable us to suggest the CICA to be an effective tool for routine monitoring of trace PV in foodstuffs. Concerning the performance time, both immunochromatographic assays were much faster than traditional ELISA techniques, indicating their promising potential for screening large numbers of samples. The fabrication cost was estimated to be ∼$0.53, with the most expensive material being anti-parvalbumin antibody (see ESM, Table S2) to enable the rapid detection of PV by the naked eye. The equipment costs, including a flatbed scanner and computer, were not included in this calculation. A more accurate estimate of the fabrication cost for semi-quantitative analysis should be calculated on the basis of the in-situ test conditions.
Conclusion Using glass capillary as the carrier, a new nano-gold immunochromatographic assay (CICA) was designed and fabricated. Using PV as the analyte, the system was optimized and its efficiency was fully evaluated and validated. In comparison to traditional immunoassays, including ELISA and NCM-based immunochromatographic techniques, the developed CICA had significant advantages, including improved sensitivity, simplicity, speed, and stability. All these results enabled us to suggest the CICA to have good potential as a new and effective technique for fast screening of biological samples.
6645 Acknowledgement This work was supported by the “Earmarked Fund for China Agriculture Research System (CARS-50)” and the Program for Changjiang Scholars and Innovative Research Team in University (IRT1188).
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