Title：Bimetallic Nanozyme: A Credible Tag for In Situ-Catalyzed Reporter Deposition in the Lateral Flow Immunoassay for Ultrasensitive Cancer Diagnosis

Journal：《Nano Letter》

IF：10.8

Original link：https://doi.org/10.1021/acs.nanolett.3c03118

Reporter：Jinrui Shan, Master of Grade 2022

In this paper, a bimetallic nano-enzyme mediated in-situ catalytic reporter deposition (BN-ISCRD) is proposed, which is designed for ultra-sensitive cancer diagnosis. The bimetallic nano-enzyme, Pd@Ir NPs, has super-high enzyme-like activity, which is further explained by density functional theory calculation, electron transfer of Pd@Ir NPs and the change of Gibbs free energy during catalysis. Taking gastric cancer biomarkers pepsinogen I and pepsinogen II as model targets, the cut-off value of this analysis can reach 10 pg/mL, which is 200 times lower than that of the analysis without signal enhancement. Eight positive and 28 negative clinical samples were correctly identified by this analysis. In a word, this LFIA based on BN-ISCRD shows great advantages and potential in the application of ultra-sensitive disease diagnosis.

Traditional colorimetric LFIA (such as LFIA based on gold nanoparticles) can be easily read by naked eyes, but its sensitivity and quantitative ability are limited, and its application in the analysis of low concentration biomarkers is limited. The combination of LFIA and new nano-materials has been used to output high-sensitivity and quantifiable signals, including fluorescence, photothermal, Raman and so on. However, these signals usually need instrument-assisted reading, which is expensive and not conducive to application in non-laboratory environment. Colorimetric signal amplification technology can improve the weak color intensity on LFIA test line (T line), and at the same time allow direct reading by naked eyes, which has attracted wide attention. However, metal reinforcing reagents used for nanoparticle growth are prone to self-nucleation, and the large labels formed by nanoparticle accumulation and assembly have low diffusion capacity and nonspecific adsorption, which limits their large-scale application. Therefore, it is necessary to develop a convenient LFIA to meet the demand of high sensitivity detection in this field.

1.Figure 1 shows the design strategy, morphology and particle size of this study, indicating the successful synthesis of Pd@Ir NPs.

Figure 1.(a) Synthesis of Pd@Ir NPs. (b and c) TEM images of Pd@Ir NPs. The scale bars are 50 and 5 nm, respectively. (d) Particle size distribution histogram of Pd@Ir NPs. (e) EDS elemental mapping images of Pd@Ir NPs. The scale bar is 5 nm. (f) XRD patterns of Pd@Ir NPs. (g and h) High-resolution XPS spectra of Pd 3d and Ir 4f, respectively.

2.Figure 2 shows Evaluation of the POD-like activity of Pd@Ir NPs.

Figure 2. (a) UV–vis spectra of TMB oxidation catalyzed by Pd NPs, Ir NPs, and Pd@Ir NPs. (b and c) Steady-state kinetic curves of Pd@Ir NPs with POD-like activity. (d) Histogram comparing the Kcat and Km values of Pd NPs, Ir NPs, and Pd@Ir NPs. (e) UV–vis absorption spectra of Cyt C and Cyt C reacted with Pd@Ir NPs. (f) Cyclic voltammetry curves of Pd@Ir NPs in the absence or presence of H2O2. (g) Relative activity of the Pd@Ir NP/TMB/H2O2 catalytic system with or without ROS scavengers. (h and i) ESR spectra of •OH and O2•–, respectively.

3.The mechanism of Pd@Ir NPs with high enzyme-like activity was studied at the atomic level by DFT calculation.

Figure 3. (a) Proposed catalytic mechanism of Pd@Ir NPs for H2O2-induced •OH production in an acidic environment. (b) Free energy diagrams of Pd@Ir NPs during catalysis. (c and d) Adsorption energy for H2O2 and reaction energy barrier of •OH dissociation from Pd NPs, Ir NPs, and Pd@Ir NPs, respectively.

4.The analytical performance of LFIA based on BN-ISCRD for biomarker detection is studied.

Figure 4. (a) Schematic representation of the BN-ISCRD-based LFIA. (b) Representative photographs taken from the strips without signal enhancement. (c and d) Curve fitting for the detection of PG I and PG II, respectively, without signal enhancement. (e) Representative photographs taken from the strips with signal enhancement. (f and g) Curve fitting for the detection of PG I and PG II, respectively, with signal enhancement. The inset shows the corresponding linear relationship between the signal intensity of the T-line and various concentrations of targets.

5.LFIA based on BN-ISCRD was used to detect PG I and PG II in serum samples collected from 36 different people.

Figure 5. (a) Procedure and time requirements for the detection of PG I and PG II in serum samples using the BN-ISCRD-based LFIA. (b and c) Heat maps showing the results of the detection of PG I and PG II, respectively, in clinical serum samples using the BN-ISCRD-based LFIA. Values represented in the heat map are the concentrations of PG I and PG II in each sample. (d and e) Correlation analysis between the BN-ISCRD-based LFIA and the turbidimetric inhibition immunoassay in quantifying the PG I concentration and PGR value, respectively, from the clinical serum samples.

In a word, a kind of LFIA based on BN-ISCRD was designed, with bimetallic nano-enzyme Pd@Ir NPs as the marker, and stronger signal was generated by in-situ catalytic chromogenic substrate deposition, which broke the limitation of traditional colorimetric LFIA in detecting ultra-low concentration biomarkers of cancer. The electron transfer ability of Pd@Ir NPs was confirmed by Cyt C electron transport experiment and electrochemical reaction. Lower adsorption energy H2O2 The lower energy barrier of OH dissociation of Pd@Ir NPs in the catalytic process was obtained by DFT calculation. The results show that the catalytic activity of Pd@Ir NPs is controlled by the thickness of Ir shell, showing a volcanic trend. The LFIA successfully realized the ultra-sensitive detection of PG I and PG II, and the COV was as low as 10 pg/mL, which was 200 times lower than that of the unreinforced one. The method realizes the simultaneous detection of two analytes in clinical samples in less than 25 min, and the results are consistent with the clinical analysis results. This BN-ISCRD strategy provides an innovative method for building an ultra-sensitive real-time detection platform, and also provides a new impetus for the combination of biosensing and new nanomaterials.