Effect of different-sized spherical gold nanoparticles grown layer by layer on the sensitivity of an immunochromatographic assay

Abstract

The immunochromatographic assay (ICA) commonly suffers from low sensitivity because of the insufficient brightness of traditional spherical gold nanoparticles (AuNSs; 20–30 nm). Larger-sized AuNSs exhibit greater potential to improve the detection performance of the ICA method because of their higher molar extinction coefficient. However, oversized AuNSs could produce steric hindrance on the strip. In the present study, the impact of AuNS size on the sensitivity of a competitive ICA method was explored. Four kinds of citrate-stabilized AuNSs with sizes of 20, 60, 100, and 180 nm were synthesized. The affinity properties of four sized AuNS probes toward ochratoxin A and bovine serum albumin conjugates were analyzed using bio-layer interferometry and further characterized by immunological kinetic analysis. These results showed that 100 nm AuNS probes could remarkably enhance the detection signal of the strip due to their higher molar extinction coefficient and stronger affinity to the target antigen. By contrast, 180 nm AuNSs were unsuitable as labeled probes on the strip because of significant steric hindrance. Under the optimal conditions, the 100 nm AuNS-based ICA sensor exhibited the best sensitivity, with a cut-off limit (qualitative detection by naked eye) and half-maximum inhibitory concentration (quantitative analysis by strip reader) in wet (dry) formats 12.5 and 7.15 (10 and 5.3) times better than those of the conventional 20 nm AuNS-based strips, respectively. In summary, 100 nm AuNSs have considerable industrial application potential as alternative novel probe nanomaterials in the competitive ICA platform.

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Introduction

The immunochromatographic assay (ICA) is one of the most popular on-site screening methods and has been widely used in clinical diagnosis, food safety, environment, and anti-terrorism monitoring.^1–6 As one of the most classical nanosized labels, colloidal gold nanoparticles (AuNSs) have occupied 90% of the label market in ICAs due to their remarkable optoelectronic properties, simple biolabeling, non-toxicity, low cost, and ease of synthesis.^7–9 Conventional spherical AuNSs (AuNSs) with a size distribution of 20–30 nm result in low ICA sensitivity because of insufficient brightness of the probe. Various novel luminescent or magnetic nanomaterials have been introduced as alternative labels to improve the analytical sensitivity of ICA because of their generation of enhanced analytical signals through excellent luminescence or magnetic properties.^10–12 In addition, several AuNS modified nanomaterials have been used to enhance ICA sensitivity because of the superior optical absorbance of the materials in comparison with pure AuNSs. Xu et al. used small AuNS (16.7 nm) modified silica nanorods (diameter = 200 nm, length = 3.4 μm) as probes for the ICA detection of proteins. The limit of detection (LOD) was 50 times lower than that of the traditional AuNS-based ICA.¹³ Tang et al. also reported AuNS decorated magnetic microspheres as novel probes for the detection of aflatoxin B₂, in which LOD is threefold lower in comparison with conventional AuNS-based ICA.¹⁴

Larger-sized AuNSs possess stronger optical absorbance in comparison with small AuNSs because of higher molar extinction coefficient of the larger AuNSs. For example, the extinction coefficient of AuNSs with a size of 80 nm is approximately 200-fold higher than that of 15 nm AuNSs.¹⁵ In theory, the use of a large-sized AuNSs as probe is beneficial to improving the sensitivity of the traditional ICA method. Laitinen and coworkers explored the size of AuNSs in range of 20 nm to 40 nm on the sensitivity of competitive ICA method, the results found that the larger size of AuNSs (39 nm) increased both maximal signals and sensitivity of the assay than smaller particles for determination of progesterone in milk.¹⁶ However, oversized probes maybe obstruct the immunological recognition between probes and detection antigen on the nitrocellulose (NC) membrane because of steric hindrance of the probe's excessively large size, and oversized AuNSs possess significantly stronger optical scattering in comparison with small AuNSs,¹⁷ thereby resulting in poor sensitivity of the ICA method. Safenkova and coworkers used five kinds of AuNSs with the size various from 6.4 to 52 nm for detection of potato virus X. The detection limit (LOD) of sandwich ICA method decreases from 80 to 3 ng mL⁻¹ with the size of AuNSs increasing from 6.4 to 33.4 nm. As for larger AuNSs with size of 52 nm, the LOD increases to 9 ng mL⁻¹.¹⁸ But, to the best of our knowledge, a comprehensive study of larger sized AuNSs up to 180 nm on the detection performances of competitive ICA method has not yet been reported.

Ochratoxin A (OTA), a secondary metabolite of Aspergillus ochraceus and Penicillium verrucosum, is one of the most extensive mycotoxin contaminants in agricultural products, such as cereals, maize, grapes, beans, nuts, coffee, cocoa, and so on.¹⁹ In 1993, the International Agency for Research on Cancer has categorized OTA as a potential human group 2B carcinogen.^20,21 Numerous countries have set strict regulations to govern the OTA level in agricultural products. In the European Union, the maximum permitted levels for OTA in raw cereals, wine, and baby food are 5, 2, and 0.5 μg kg⁻¹, respectively.^22–24 The World Health Organization has set a tolerable weekly intake of 100 ng kg⁻¹ of body weight as well.^25,26

To explore the influence of AuNS size on the detection performance of the competitive strip, we synthesized four kinds of AuNSs with sizes of 20, 60, 100, and 180 nm, respectively. OTA was selected as a model analyte, and unpurified anti-OTA ascitic fluids were used to label four kinds of AuNSs to prepare AuNS probes. The effects of AuNS diameter on the binding properties of probes were analyzed by determination of the association rate (k_a), the dissociation rate (k_d), and the binding affinity (K_D) of AuNS probes on OTA–bovine serum albumin (BSA) antigens. Meanwhile, immunological dynamic characterizations of four kinds of AuNS probes on the NC membrane were also evaluated. Furthermore, the effect of AuNS size on the detection performance of competitive ICA was confirmed by comparison of the cut-off limit of strips by the naked eye, the half maximal inhibitory concentration (IC₅₀), LOD for quantitative analysis, and AuNS consumption for each strip.

Material and methods

Materials and reagents

HAuCl₄·3H₂O, citric acid, hydroquinone, biotin-3-sulfo-N-hydroxysuccinimide ester sodium salt (C₂₀H₃₀N₄O₆S), BSA, and OTA were purchased from Sigma-Aldrich (St. Louis, MO, USA). The OTA standard solution was prepared as a 1 mg mL⁻¹ solution in methanol and working dilution by deionized water. The unpurified anti-OTA ascitic fluids were provided by Wuxi Zodoboer Biotech. Co., Ltd. (Wuxi, China). The sample pad, NC membrane (CN140), and absorbent pad were obtained from Schleicher and Schuell GmbH (Dassel, Germany). Phosphate-buffered saline (PBS, 0.1 M) was prepared by adding 12.2 g K₂HPO₄, 1.36 g KH₂PO₄, and 8.5 g NaCl into 1000 mL Milli-Q water and adjusted to pH 7.4 (unless otherwise specified) prior to use. Ultra-pure water was prepared by Milli-QA (Molsheim, France). All other reagents were of analytical grade and purchased from Sinopharm Chemical Corp. (Shanghai, China).

Synthesis of different sizes of gold nanoparticles

Small AuNSs with diameter of 20 nm were prepared by a typical citrate reduction method with slight modification.²⁷ Briefly, 100 mL of 0.01% (w/v) HAuCl₄ solution was heated to boiling and 2.7 mL of 1% (w/v) sodium citrate solution was quickly added under constant stirring, and maintained at the boiling temperature for an additional 10 min. The concentration of the obtained 20 nm AuNS solution was calculated at 1.17 nmol L⁻¹ according to the methods of Haiss.²⁸ AuNSs with diameters of 60, 100 and 180 nm were synthesized following a kinetically controlled seeded growth strategy according to a previous report with some modifications.²⁹ Among these particles, AuNSs with a size of 60 nm were prepared by adding 1.0 mL of as-prepared 20 nm AuNS and 800 μL of 1% HAuCl₄ solution to 100 mL deionized water under vigorous stirring. Subsequently, 200 μL of 1% trisodium citrate and 100 μL of hydroquinone (30 mM) solutions were added every 10 min for five times. At the same concentration of gold seeds, larger AuNSs with sizes of 100 and 180 nm were synthesized by adding 2.4 and 4 mL of 1% HAuCl₄ solutions to 100 mL deionized water, respectively. Under vigorous stirring, 400 μL of 1% trisodium citrate and 200 μL of hydroquinone (30 mM) solutions were added every 10 min, and the addition of the two solutions were repeated for 8 and 12 times, respectively. After continuous reaction for another 1 h, the three obtained AuNS solutions were centrifuged, and pellets were re-dissolved in 10 mL of 0.02% (w/v) sodium citrate solution and stored at 4 °C for further use. The concentrations of 60, 100 and 180 nm AuNS solutions obtained by seed-growth method were 0.117 nmol L⁻¹ based on the amounts of added gold seeds.

The size and morphology of four different AuNSs were determined using transmission electron microscopy (TEM, JEM-2100HR, JEOL Ltd., Japan). Surface plasma resonance (SPR) absorption spectra were recorded using a UV-vis spectrophotometer (Shimadzu, UV-2300, Japan). The hydrodynamic diameter and monodispersity of the obtained AuNSs were characterized using a particle size analyzer (Malvern Instruments Ltd., Worcestershire, U.K.).

Preparation of four AuNS probes

The unpurified anti-OTA ascitic fluids were used to label four different AuNS solutions according to the previous reports.³⁰ First, the pH levels of the four AuNS solutions were adjusted to 6.5 with 0.2 mol L⁻¹ K₂CO₃. Then, 14.21 μg of ascites were added dropwise to 1 mL of 20 nm AuNS solution (1.0 nmol L⁻¹), whereas 5.62, 11.84, and 25.64 μg of anti-OTA ascites were added to 1 mL of AuNS solutions (10 pmol L⁻¹) with the sizes of 60, 100, and 180 nm, respectively. After being gently stirred for 60 min, the solution was added with 100 μL of 1% (w/v) PEG 20000 solution and blocked for another 60 min. The mixtures were centrifuged, and precipitates were resuspended in 50 μL of multiplexed solution containing 2% fructose, 1% PEG 20000, 5% sucrose, 1% BSA, and 0.4% Tween-20. Four different AuNS probes were stored at 4 °C for further use.

Measurements of the affinities of four AuNS probes

The affinities of four AuNS probes to OTA–BSA conjugates were determined using bio-layer interferometry (BLI; ForteBio, Menlo Park, CA, USA). Biotin-labeled OTA–BSA conjugates (biotin-BSA–OTA) were obtained by coupling the carboxy group of biotin-3-sulfo-N-hydroxysuccinimide ester sodium salt to the amino group of OTA–BSA. In a typical procedure, 10 μL of biotin-3-sulfo-N-hydroxysuccinimide ester sodium salt solution (100 nmol L⁻¹ in DMF) was added to 200 μL of BSA–OTA solution (5 nmol L⁻¹ in pH 8.0, 0.01 mol L⁻¹ PBS buffer). After 45 min of reaction, the solution was dialyzed in 0.01 mol L⁻¹ PBS (pH 7.4) buffer for 72 h to remove free biotin. Then, the kinetic assay of four AuNS probes to OTA–BSA antigen was conducted according to the following procedure. First, streptavidin biosensor was prewetted and equilibrated with PBS buffer (0.01 mol L⁻¹, pH 7.4). Second, 4 μL of biotin-BSA–OTA (0.16 mg mL⁻¹) was non-covalently loaded on the surface of streptavidin biosensor by an additional incubation time of 600 s. After a 300 s equilibration step, association assay of each AuNS probe to OTA–BSA antigen was conducted for 600 s with four different concentrations in PBS buffer. The four concentrations for 20 nm AuNS probe were 1.174, 0.587, 0.391, and 0.294 nmol L⁻¹, respectively, whereas those for the other three sizes of probes were 11.74, 5.87, 3.91, and 2.94 pmol L⁻¹, respectively. Then, dissociation assays were carried out with PBS for 300 s. All measurements were performed in triplicates. Finally, the association rate (k_a), dissociation rate (k_d), and equilibrium dissociation constants (K_D) of four AuNS probes were obtained by loading experimental data into the Octet Data Analysis software.^31,32

Immunological kinetic analysis of different sized AuNS probes on the strip

Immuno-dynamic analysis between AuNS probes and antigen interaction (OTA–BSA on the T line) on the strip was performed according to our previous report.^33,34 In a typical procedure, certain amounts of AuNS probes were premixed with 75 μL of PB buffer for 3 min, and the mixtures were pipetted into the sample well. So as to guarantee sufficient color intensity on the test line, the contents of 20 nm AuNS probes were ten-fold that of the three other kinds of AuNS probes. After running for 1 min, the optical densities of the strip on the test (OD_T) and the ratio of OD_T/OD_C (T/C) were recorded by a commercial HG-8 strip reader (Shanghai Huguo Science Instrument Co., Ltd., Shanghai, China) every 30 s for 25 min. Kinetic curves were established by plotting the OD_T, and T/C values against time.

Performance evaluation of different sized AuNS-based strips

The preparation of the strips was conducted according to some previous report.^30,35 As shown in Scheme 1, the strip comprised four parts, namely, the sample pad, the conjugate pad, the absorbent pad, and the NC membrane. The sample pad was pretreated with 50 mM of borate buffer (pH 7.4) containing 1% BSA, 0.5% Tween-20, and 0.05% sodium azide, and further dried at 60 °C for 2 h. Glass fiber membrane, which was pretreated with 0.01 M PBS (pH 7.4) solution containing 0.2% Tween-20, and 2% sucrose at 60 °C for 4 h, was used as conjugate pad. Absorption pad was used without further treatment. Certain concentrations of OTA–BSA (1.32 mg mL⁻¹) and donkey anti-mouse IgG (0.25 mg mL⁻¹) were sprayed on the NC membrane as the test (T) and control (C) lines, respectively, with a dispensing density of 0.75 μL cm⁻¹. The distance between T and C lines was 6 mm. The NC membrane was then dried at 37 °C for 12 h.

Scheme 1 Schematic of the different-sized AuNSs based strips for OTA detection.

Strip tests, including wet and dry formats, were conducted to explore the size of AuNSs on ICA sensitivity. For the wet test,³⁴ certain amounts (4.7, 0.205, 0.1175, and 0.45 fmol) of AuNS probes with sizes of 20, 60, 100, and 180 nm, respectively, were premixed with 75 μL of OTA sample solutions in the tube for 5 min and then added in the sample well of the strip. For the dry test format,³⁶ two kinds of AuNS probes with concentrations of 4.7 (20 nm) and 0.1175 nmol L⁻¹ (100 nm) were jetted onto the conjugate pad at a density of 3.0 μL cm⁻¹. The as-prepared conjugate pads were dried at 37 °C for 3 h with a vacuum dryer, and then laminated with the NC membrane, the sample pad, and the absorbent pad. The effects of AuNS size on strip sensitivity were evaluated by comparison of IC₅₀, LODs of the strips for quantitative analysis, and cut-off values for qualitative detection. OTA standard solutions were prepared by adding an OTA stock solution (2.0 μg mL⁻¹) into PBS buffer containing 10% methanol to OTA final concentrations of 0 (as a negative control), 0.05, 0.1, 0.2, 0.3, 0.5, 0.8, 1, 1.5, 2, 2.5, 3, 4, 5, 8, 10, 15, 20, 25, 30, and 40 ng mL⁻¹, respectively. The quantitative curves of strips were established by plotting B/B₀ against the logarithm of the OTA concentrations according to our previous report,³⁷ B₀ and B represent the ratio of T/C of the negative sample and an OTA spiked standard solutions, respectively. IC₅₀ values were obtained from five independent experiments. The LODs of strips were defined as 10% of OTA competitive inhibition concentration, whereas cut-off values for qualitative analysis through the naked eye were defined as the minimum concentration of OTA that does not cause red color on the T line after 10 min of incubation time.

Results and discussion

Synthesis and characterization of different sizes of AuNSs

In the present study, 20 nm-sized AuNSs were synthesized by using a citrate reduction method. Larger AuNSs with sizes up to 180 nm were synthesized according to a hydroquinone reduction method with certain modification.^38,39 In the method, a kinetically controlled seeded growth strategy was introduced to produce a series of larger-sized AuNSs with a narrow size distribution. The diameter of the AuNSs was adjusted by changing the numbers of gold growth round, whereas the shape of AuNSs was regulated by controlling the concentrations of hydroquinone, HAuCl₄, and sodium citrate in the reaction system. The UV-vis spectra of four kinds of AuNSs (Fig. 1A) show that the maximum plasma resonance absorption (SPR) peaks are 520, 547, 586, and 611 nm, respectively. Meanwhile, the optical absorbance of resultant AuNSs shows significant enhancement with the increase in size of AuNSs from 20 nm to 100 nm at the same particle concentration. However, the absorbance of 180 nm AuNSs appears a sharply decline because of its strong light scattering.^40–42 Fig. 1B shows that the average hydrodynamic diameters of as-prepared AuNSs were 21 ± 0.3, 62 ± 0.7, 105 ± 0.6, and 187 nm ± 1.4 nm, respectively, whereas polydispersity indices (PDI) of four AuNS solutions were 0.23, 0.19, 0.18, and 0.13, indicating that the obtained AuNSs possess good monodispersity with a narrow size distribution. TEM observation in Fig. 1C reveals that the average sizes of four AuNSs were 20 ± 1, 60 ± 3, 100 ± 5, and 180 nm ± 6 nm (n = 50). The four kinds of AuNSs did not exhibit flocculation or aggregation and could maintain good colloidal stability in solution.

Fig. 1 Characterization of different-sized AuNSs. (A) UV-visible spectra of different-sized AuNSs; (B) hydration diameter and polydispersity index of different-sized AuNSs; (C) TEM images and physical photos of different-sized AuNSs (scale bars are 50 nm in the TEM images).

Affinity properties of four kinds of AuNS probes

The affinity properties of AuNS probes are one of the most important parameters that influence the sensitivity of the ICA method. To elaborate the effect of AuNS size on the affinity of AuNS probes, we labeled different AuNSs with sizes of 20, 60, 100, and 180 nm with a saturated amount of anti-OTA ascitics to produce the same density of monoclonal antibodies (mAbs) on the surface of AuNSs. In our previous study, we have comfirmed that labeled probe using unpurified ascitics instead of purified mAbs could retain more bioactivity of probes, since it doesn't suffer from a series of complex procedures for mAbs purification.^40–42 The pH levels of four kinds of AuNS solutions were adjusted by adding 0.2 mol L⁻¹ K₂CO₃ to a final pH of 6.5 for labeling anti-OTA ascitics. The optimization of saturated labeled amounts of anti-OTA ascitics on different-sized AuNSs was presented in Fig. S1,† indicating that the saturated ascitic amounts of 20, 60, 100, and 180 nm AuNSs were 0.0142, 0.562, 1.184, and 2.564 μg ascitics per fmol of AuNSs, respectively. These results show that the protein loading capacities of AuNSs with sizes of 60, 100, and 180 nm are approximately 40-fold, 83-fold, and 181-fold higher than that of 20 nm AuNSs due to their larger surface area, respectively, which is in accordance with Laitinen's report.¹⁶ The affinity properties of the above AuNS probes were determined by using BLI. The kinetic binding curves between the OTA–BSA antigen and the AuNS probes are shown in Fig. S2.† The affinity properties of the four kinds of AuNS probes to OTA–BSA antigen, including k_a, k_d, and the K_D constant, are summarized in Table 1. Results show that the K_D of AuNS probe significantly decreased from 6.0 × 10⁻¹¹ M to 6.4 × 10⁻¹² M with the increase in AuNS size from 20 nm to 180 nm, indicating a stronger binding ability of the larger AuNS probe to the OTA–BSA antigen. Safenkova et al. found that the increase in AuNS size in the range from 5 nm to 60 nm could lead to an increase in the affinity of mAbs and AuNS conjugates.⁴³ Our results also demonstrate a similar conclusion even if the size of AuNSs increases to 180 nm. Simultaneously, we also found that the k_d values of 60, 100, and 180 nm AuNS probes all exceed that of 20 nm AuNS probe, and the maximum k_d value of 100 nm AuNS probe is approximately 25.5-fold higher than that of conventional 20 nm AuNS probe. The larger k_d value between the competitive antigen and the probe favors the improvement of the detection performance of the competitive ICA method. In consequence, 100 nm AuNS-based ICA strips may exhibit higher sensitivity for the detection of mycotoxins.

Table 1 Characterization of affinity parameters for different sized AuNS probes and OTA–BSA artificial antigen using BLItz® system

Diameter of gold nanoparticles (nm)	KD (M)	ka (M^−1 s^−1)	kd (s^−1)
20	6.004 × 10^−11	6.178 × 10^5	3.709 × 10^−5
60	4.721 × 10^−11	1.095 × 10^7	5.168 × 10^−4
100	4.424 × 10^−11	2.138 × 10^7	9.457 × 10^−4
180	6.435 × 10^−12	3.507 × 10^7	2.257 × 10^−4

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Immunological kinetics analysis of four kinds of AuNS probes on the strip

In the ICA sensor, AuNSs perform two important functions, including a recognition probe function to react with the target analyte and as a signal element to generate a detection signal. For the strip assay, oversized AuNSs (e.g. 180 nm) as probe could exhibit obvious steric hindrance between antibody on the probes and antigen on the NC membrane, thereby resulting in a weak color on the T or C lines.^44,45 To explore the size of AuNSs on the detection performance of the competitive ICA method, the immuno-dynamic process of the AuNS probe and antigen interaction on the strip was carried out by recording of OD_T and T/C values against running time in every 30 s for 25 min because the development of OD values on T line can indirectly reflect the antibody–antigen dynamic interactions.^46,47 The contents of 60, 100, and 180 nm AuNS probes for each strip assay were 0.47 fmol, whereas the amount of 20 nm AuNS probes (4.7 fmol) was tenfold that of the other three sizes of AuNS probes because of inadequate brightness. Kinetic curves on the test line are shown in Fig. 2A, indicating that the OD_T values of four kinds of probes increase sharply in the first 10 min of immunoreaction and then ascend slowly during the 10–20 min analysis time, ultimately reaching a dynamic balance. Among probes, the OD_T of 100 nm AuNS probes was achieved at the maximum value of 824 ± 25 after 20 min of incubation time. This value is 2.4-fold higher than that of the 20 nm AuNS probe, although the amount of 100 nm AuNS probes was tenfold less than that of 20 nm AuNS probes. The OD_T of 180 nm AuNS probes on the strip was only 443 ± 13, which was 1.9-fold lower than that of 100 nm AuNS probe. These results indicated that a steric hindrance of 180 nm AuNSs could reduce the optical absorbance of T line on the strip. In addition, Fig. 2B shows that the T/C ratios of four kinds of AuNS probes could more quickly reach a constant value than the corresponding OD_T value. These results indicate that the ratio of T/C value could shorten the interpretation time of ICA quantitative analysis. Moreover, Fig. 2B also shows that the T/C ratio of the 100 nm AuNS probe is remarkably higher than those of the three other kinds of AuNS probes. In theory, high-affinity probes could produce higher T/C values because probes are more easily captured by the antigen on the test line. Although the affinity of 180 nm AuNS probe is significantly higher than that of 100 nm AuNS probe (Table 1), the T/C ratio of 180 nm AuNS probes (0.510 ± 0.013) on the strip is markedly below that of 100 nm AuNS probes (0.850 ± 0.022) and even less than that of 60 nm AuNS probes (0.70 ± 0.016). The above results demonstrate that oversized AuNSs (e.g. 180 nm) are unsuitable for labeling the probe on the strip because of the significant steric hindrance, and 100 nm AuNSs were considered as excellent colored nanomaterials to substitute conventional 20 nm AuNSs for the competitive strip assay. To further confirm the advantages of the 100 nm AuNSs on the strip assay, we suggested a series of concentrations of different-sized AuNS probes for running the strips under the same experimental conditions. The OD_T and T/C values of all strips were recorded at a 25 min assay time. The results in Fig. 2C and D demonstrate that the 100 nm AuNS probes could produce the highest OD_T and T/C values under a series of AuNS probe concentrations. The stereogram of strips (Fig. S3†) of various-sized AuNS probes with the same amount (0.235 fmol) further verifies that the visual color of the 100 nm AuNS-based strip on the T line is significantly stronger than that of the three other-sized AuNS-based strips, and OD_T value of 629 ± 6 was achieved (n = 3); whereas the 20 nm AuNS-based strip using the same amount probe showed virtually almost no color bands on both lines because of its inadequate brightness. The results in Fig. 2C also indicate that an approximate 11.75 fmol of conventional 20 nm AuNS probes could generate basically the same color on the test line (614 ± 8, n = 3), in which the amount of 20 nm AuNS was 50-fold that of 100 nm AuNS probes. In summary, the above results verify that 100 nm AuNSs as probe carriers can significantly enhance the visual color on the test line and decrease the dosage of probes on the strip because of higher molar-extinction coefficient and stronger affinity to the target antigen.

Fig. 2 Immunoreaction dynamic analysis of AuNS-based ICA. (A) Immunoreaction dynamic curve of the test line (OD_T) against immunoreaction time with different-sized AuNS probes; (B) immunoreaction dynamic curves of T/C against immunoreaction time with different-sized AuNS probes; (C) OD_T values of four kinds of strips with various concentrations of AuNS probes; (D) T/C values of four kinds of strips with various concentrations of AuNS probes. *In addition, in (A and B), the amounts of AuNS probes for each strip were 4.7 fmol for 20 nm AuNSs and 0.47 fmol for 60, 100, and 180 nm AuNSs, respectively; in (C and D), the concentrations of 20 nm AuNS probes is tenfold that of the abscissa values in the figure.

Performance comparison of four sizes of AuNS-based strips

To verify whether 100 nm AuNSs could improve the sensitivity of the ICA method, four kinds of AuNS probes were used to run the strips. Strip tests include wet (preincubation probe and sample solution in tube) and dry (sprayed probe on the conjugate pad) formats. In this study, we first used the wet format to evaluate AuNS size on the sensitivity of the strip. To reduce the influence of color variation of the test line on the sensitivity of the strip, the OD_T value of four kinds of strips were required to control basically the same level at approximately 400 for the detection of the OTA free sample. Among the strips, the parameters of 20 nm AuNS-based strip were optimized by a similar “checkerboard titration” method with different amounts of AuNS probes under various concentrations of BSA–OTA conjugates on the T line for various combinations. The results shown in Table S1† indicate that the optimal combinations were 1.32 mg mL⁻¹ of OTA–BSA and 0.25 mg mL⁻¹ of donkey anti-mouse IgG sprayed on the NC membrane as the T and C lines, respectively, and 4.7 fmol of 20 nm AuNS probes used for running the strip. Under optimal conditions, the strip exhibited two clear red bands on test and control zones, in which the means of OD_T and OD_C for the detection of OTA negative sample are 416.1 ± 3.3 and 353.9 ± 4.8, respectively. The competitive inhibition rate for 2 ng mL⁻¹ OTA spiked sample was the highest (44.29%) among the studied samples. The other three kinds of strips were prepared by altering the amounts of 60, 100, and 180 nm AuNS probes, whereas the concentrations of OTA–BSA and donkey anti-mouse IgG on the NC membrane were the same as that of 20 nm AuNS-based strip. The results show that only 0.205, 0.1175, and 0.45 fmol of 60, 100, and 180 nm AuNS probes, respectively, were sufficient to achieve nearly identical signal intensities on the T line with the 20 nm AuNS-based strip. The detection performances, including naked-eye based qualitative detection and strip reader-based quantitative analysis, of four kinds of strips were determined. In the qualitative detection by ocular inspection, the cut-off limit of 20 nm AuNS-based strip was 25 ng mL⁻¹, whereas those of 60, 100, and 180 nm AuNS-based strips were 2.5, 2.0, and 5.0 ng mL⁻¹, respectively. In particular, the comparative stereogram of 20 and 100 nm AuNS-based ICAS for the detection of different concentrations of OTA are presented in Fig. S4.† For strip quantitative analysis, the calibration curves of four kinds of strips were constructed by plotting B/B₀ against the logarithm of various concentrations of the OTA analytical standard (0–25 ng mL⁻¹). The regression equation of 20 nm AuNS-based strip is y = 0.181 ln x + 0.542 (R² = 0.998), where y is the ratio of B/B₀ and x is the OTA concentrations from 0 ng mL⁻¹ to 25 ng mL⁻¹. IC₅₀ was 1.93 ng mL⁻¹ (n = 3), and the LOD was 0.909 ng mL⁻¹ according to 10% OTA competitive inhibition concentration. The other regression equations of 60, 100, and 180 nm AuNS-based strips are shown in Fig. 3, and the IC₅₀ values of 60, 100, and 180 nm AuNS-based strips were calculated as 0.62, 0.27, and 2.24 ng mL⁻¹ according to regression equations. The LODs of three other kinds of strips were 0.034, 0.025, and 0.459 ng mL⁻¹, respectively. The above results indicate that the cut-off limit and IC₅₀ value of 100 nm AuNSs as strip probe are significantly lower (12.5-fold and 7.2-fold, respectively) than that of conventional 20 nm AuNSs, and the amount of 100 nm AuNS probes are 40-fold lower than that of conventional 20 nm AuNSs for each strip.

Fig. 3 Calibration curves of different-sized AuNS-based strips in wet format. (A) 20 nm AuNSs; (B) 60 nm AuNSs; (C) 100 nm AuNSs; and (D) 180 nm AuNSs.

The strip with a dry format is the most popular commercial product. Therefore, we further evaluate 20 and 100 nm AuNS probes on the detection performance of the strip with the dry method. The concentrations of OTA–BSA and donkey anti-mouse IgG on the strip were the same as those of the wet format. To achieve the OD_T value (approximately 400) that is consistent with the wet format strip, the spraying amounts of 20 and 100 nm AuNS probes on conjugate pads were adjusted at 14.1 and 0.353 fmol cm⁻¹, respectively, which are approximately 1.2-fold of the wet method. The results shown in Fig. 4 indicate that the cut-off limit and IC₅₀ value of 100 nm AuNS-based ICA were 0.48 and 2.5 ng mL⁻¹, respectively, which were tenfold and 5.3-fold better than that of conventional 20 nm AuNS-based strips in the dry format. The above results further confirm that 100 nm AuNSs were alternative novel probe nanomaterials for improving the sensitivity of the competitive ICA method.

Fig. 4 Calibration curves of different-sized AuNS-based strips in dry format. (A) 20 nm AuNSs; (B) 100 nm AuNSs.

Conclusions

We demonstrated that the size of AuNSs can significantly influence the detection performance of ICA method in two aspects, including the color development and the affinity of the AuNS probe. Larger-sized AuNSs (e.g. 100 nm) as probe carrier can notably enhance the OD_T value of the strip because of higher molar extinction coefficient and stronger affinity to the target antigen. However, oversized AuNSs (e.g. 180 nm) could result in poor color on the test zone because of significant steric hindrance and strong light scattering. Using 100 nm AuNSs as strip probe carriers, the IC₅₀ and cut-off limit of the strips were 7.15 and 12.5 times in wet format, 5.3 and 10 times in dry format better than those of the conventional 20 nm AuNSs based strips, respectively. In summary, larger-sized AuNSs as an alternative labeling nanomaterials exhibit significant advantages in improving the sensitivity of the competitive ICA method, also providing a promising platform for highly sensitive point-of-care test.

Acknowledgements

This work was supported in part by the National Basic Research Program of China (2013CB127804), Training Plan for the Main Subject of Academic Leaders of Jiangxi Province (20142BCB22004).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c6ra03695c

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