Avoiding the self-nucleation interference: a pH-regulated gold in situ growth strategy to enable ultrasensitive immunochromatographic diagnostics

Background: Gold nanoparticle-based immunochromatographic assay (AuNP-ICA) has insufficient sensitivity due to its inherent colorimetric signal intensity and low capture efficiency of AuNPs. The metal in situ growth is a common strategy to enhance the sensitivity of AuNP-ICA due to its superior signal amplification potential and simple operation. However, the detection distortion caused by metal self-nucleation during the growth process can seriously affect the accuracy and reproducibility of the strips. Methods: We present a pH-regulated gold in situ growth (GISG) strategy to amplify the colorimetric signal and demonstrate its application in improving the performance of traditional AuNP-ICA. The controllable growth signal amplification is achieved by lowering the pH of the growth solution to weaken the reducibility of hydroxylamine (HA), thus urging the crystallization and growth of Au3+ on the AuNP surface instead of free reduction and self-nucleation. In addition, the mechanism of pH regulation on HA reducibility is elucidated by introducing an electron-donating or electron-withdrawing group to affect the electron density of hydroxyl group. Results: The proposed GISG strategy shows improved sensitivity, low background, robust operation, and good reproducibility. The LOD values of the designed GISG-amplified AuNP-ICA are as low as 0.0198 ng mL-1 for hepatitis B surface antigen and 0.0125 ng mL-1 for HIV-1 capsid p24 antigen, which are lower by about 500- and 70-fold, respectively, than those of the unamplified AuNP-ICA. Conclusions: This method is extended to enable ultrasensitive and rapid diagnosis of viral infections, and has potential as a general signal amplification platform to redefine immunochromatographic diagnostics.


Experimental procedures S1 Materials
Hydrogen tetrachloroaurate (III) trihydrate (HAuCl 4 •3H 2  was prepared by adding 35.8 g Na 2 HPO 4 ·12H 2 O and 15.6 g NaH 2 PO 4 ·2H 2 O in 1000 mL Milli-Q water. pH was adjusted to 7.4 before use unless otherwise specified. All other analytical-grade chemicals were bought from Sinopharm Chemical Corp. (Shanghai, China) and used without further purification.

S2 Instrumentation
UV-visible absorption spectra were obtained on a UV-vis spectrophotometer

S3 Synthesis of 18 nm citrate-coated AuNPs
Citrate-coated AuNPs were synthesized using the citrate reduction method described in our previous work. [1] In a typical synthesis, 2.7 mL of 1% sodium citrate solution was added to 100 mL of boiling 0.01% gold chloride trihydrate solution under constant stirring. Citrate-coated AuNPs (18 nm) were obtained after 10 min of reaction when the color of the solution changed from light yellow to red purple.

S4 Preparation of anti-p24 dAbs@AuNP probes
The probes of p24 were prepared by immobilizing anti-p24 dAbs on the surface of citrate-coated AuNP via electrostatic adsorption. [2] Typically, the pH of 1 mL citrate-coated AuNP solution was adjusted to 8.5 with 0.01 M K 2 CO 3 , added dropwise with 5 µg anti-p24 dAbs solution after 1 h incubation under gentle stirring at 25 °C, and added with 500 μL of 10% BSA (w/v). The mixed solution was incubated for another 60 min. The as-prepared anti-p24 dAbs@AuNP was then purified via centrifugation and resuspended in 0.01 M PB buffer (pH = 7.4) containing 25% sucrose and 0.1% sodium azide and stored at 4 °C until further use.

S5 Preparation of anti-HBsAg dAbs@AuNP probes
The probes of HBsAg were prepared by immobilizing anti-HBsAg dAbs on the surface of the citrate-coated AuNP via electrostatic adsorption. [2] Typically, the pH of containing 25% sucrose and 0.1% sodium azide and stored at 4 °C until further use.

S6 Fabrication of GISG-amplified AuNP based test strip
Similar to traditional AuNP-based test strip, the proposed GISG-amplified AuNP S4 based test strip were composed of the following parts: a sample pad, NC membrane, and absorbent pad. Anti-HBsAg/p24 cAbs (1.0 mg mL −1 ) and goat antimouse IgG (1.0 mg mL −1 ) prediluted in 0.01 M PB buffer (pH = 6.0) were spotted onto NC membranes as a test (T) line and a control (C) line at a distance of 4 mm. The sample pad, NC membrane, and absorption pad were then assembled into a plastic backing plate, cut into 4 mm wide strips, and packaged in a plastic casing for subsequent storage in a drying cylinder at room temperature.

S7 HBsAg detection through GISG-amplified AuNP-ICA
In this experiment, 2 µL as-prepared detection probes (anti-HBsAg dAbs@AuNP, and C (OD C ) lines in each cycle of the AuNP assembly were recorded using the commercial HG-8 strip reader, and the corresponding photograph of the reacted strips was obtained using a digital camera (Sony DX3400, Tokyo, Japan).