Bionic E-Eye Based High-Throughput Immunocolorimetric Sensor System for Sensitive Detection BNP in Blood through Carbon-Gold Nanocomposites
Authors: Liu Xin , Gan Ying , Li Fengheng , Qiu Yong , Jiang Deming , Kong Liubing , Zhu Yuxuan , Liang Tao , Zhou Shuqi , Wan Hao , Wang Ping 
Published: July 2021

Introduction

Precise and sensitive detection of protein biomarkers is of vital importance in clinical diagnosis and biomedical research. To date, enzyme-linked immunosorbent assay (ELISA) remains the gold standard in detecting protein biomarkers due to the robust properties of simple operation, acceptable sensitivity, and automated high throughput [1, 2]. Despite desirable results from ELSA, pursuing high sensitivity and accuracy to meet diagnosis demands is still an ongoing endeavor. For that, nanomaterials behaving with favorable physical properties and biocompatibility may serve as a solution, especially the carbon nanosphere (CN) or gold nanoparticles (AuNPs) that feature strong stability, easy manufacture, low cost, and environment-friendly, and abundant active sites [3, 4].
Heart failure (HF) is one of the most threatening cardiovascular diseases. B-type natriuretic peptide (BNP), a vital cardiac biomarker, has been acknowledged as one of the principal biomarkers for HF [5]. Individuals at risk of HF demonstrate raised BNP in serum and real-time quantification of HF biomarkers, identifying those at highest risk of HF early, and ensuring they receive appropriate treatment can effectively prevent premature death [6]. Although current ELISA-methods for detecting BNP have been developed, there are urgent needs for providing more efficiently and reliably in vitro diagnostic (IVD) methods or point-of-care (POC) devices in BNP clinical measurement.
Most of the previous studies for ELISA-based BNP detection required large instruments with high costs such as microplate readers, spectrophotometers. In this work, we adopted a self-developed portable bionic electronic eye (Bionic E-eye) system using a smartphone (iPhone 4S and iPad 3) integrated with simple accessories including a piece of electroluminescent, a wide-angle lens, and a dark hood instead of the commercial microplate reader to make the entire analysis process more intelligent and convenient. In addition, the CN-AuNPs nanocomposites (CGN) we prepared was used as a signal amplifier to develop a high-throughput immunocolorimetric method that sensitively identified BNP through the color change of antibody-antigen reactions using enzyme-substrate and horseradish peroxidase-antibody (pAb-HRP) linked CGN immunoprobe (CGNs@ pAb-HRP). The combination of Bionic E-eye and high-throughput immunocolorimetric system aimed to establish a general and reliable strategy for POC testing (Figure. 1A).

Method

Monoclonal antibody against BNP were immobilized on the surface of the 96-well plate as capture antibody. Then the different concentrations of BNP were added to the wells. Finally, the HRP-labeled polyclonal BNP (pAb-HRP) antibody or the pAb-HRP modified CGNs (pAb-HRP-CGNs) were added to bind with BNP. After 3,3',5,5'-Tetramethylbenzidine (TMB) and HCl were added, different shades of yellow color related to BNP concentration appeared. Combining with the Bionic E-eye, BNP concentration could be analyzed by image processing.

Results and Conclusions

To achieve optimally sensitive signal output and fabricate a well-performance immunoprobe, the synthesized conditions were optimized. Under optimal conditions, we have successfully synthesized stable and homogeneous CGNs validated by TEM (Figure. 1B) and XPS. The performance for BNP detection by a commercial microplate reader (Figure. 1C) and Bionic E-eye were evaluated and compared. The detection range of both was comparable (7.8 ppb~125 ppb) while the Bionic E-eye system had a lower limit of detection (LOD) than a commercial microplate reader (4.3 ppb < 7.2 ppb) (Figure. 1D), indicating that compared with a commercial colorimetric reader the self-developed system achieved higher sensitivity which could be attributed to the superiority of saturation channel in HSV or RGB color model the Bionic e-Eye used. In addition, the signal amplifying effect of the pAb-HRP-CGNs probe was also confirmed in the comparison of LOD and detection range with pAb-HRP. Figure. 1E showed that the LOD in the method using pAb-HRP-CGNs reached 0.92 ppb and the wider detection range of 3.9 ppb~250 ppb was achieved compared with that of 7.8 ppb~125 ppb in the non-CGNs method. Based on the above results, this proposed CGNs based detection of BNP with Bionic e-Eye shows promising application prospect but more experimental optimizations should be performed to further improve its performance.

References

[1] Shi, C., et al., Nanoscale technologies in highly sensitive diagnosis of cardiovascular diseases. Frontiers in Bioengineering and Biotechnology, 2020. 8.
[2] Jiao, L., et al., Au@Pt nanodendrites enhanced multimodal enzyme-linked immunosorbent assay. Nanoscale, 2019. 11(18): p. 8798-8802.
[3] Xu, T., et al., Triple tumor markers assay based on carbon–gold nanocomposite. Biosensors and Bioelectronics, 2015. 70: p. 161-166.
[4] Zeng, S., et al., A review on functionalized gold nanoparticles for biosensing applications. Plasmonics, 2011. 6(3): p. 491-506.
[5] Szunerits, S., et al., Electrochemical cardiovascular platforms: current state of the art and beyond. Biosensors and Bioelectronics, 2019. 131: p. 287-298.
[6] Savonnet, M., et al., Recent advances in cardiac biomarkers detection: From commercial devices to emerging technologies. Journal of Pharmaceutical and Biomedical Analysis, 2020: p. 113777.

Figure 1