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Enhanced nanobody-driven bioluminescent immunoassay for rapid parathion detection using engineered
Yi-Fan Liang1, Qiu-Yue Pan1, Yu Wang2
1Guangdong Provincial Key Laboratory of Food Quality and Safety, College of Food Science, South China Agricultural University, Guangzhou, Guangdong, 510642, China.
Biosensors & Bioelectronics
|November 16, 2024
Summary
A new nanobody-driven bioluminescent immunoassay using engineered split-nanoluciferase (NanoLuc) offers enhanced detection of the organophosphate pesticide parathion. This NanoLuc Ternary Technology (NanoTeT) system provides a faster, more sensitive, and reliable method for detecting small-molecule contaminants.
Area of Science:
- Biotechnology
- Analytical Chemistry
- Immunotechnology
Background:
- Organophosphate pesticides, like parathion, pose significant risks to public health and the environment.
- Existing immunoassay methods for small-molecule contaminants often lack sufficient sensitivity, speed, or simplicity.
- Nanobody-based assays and split-luciferase systems offer potential for developing highly sensitive and rapid detection platforms.
Purpose of the Study:
- To develop an enhanced nanobody-driven bioluminescent immunoassay for the sensitive detection of parathion.
- To establish a novel NanoLuc Ternary Technology (NanoTeT) system for improved signal-to-noise ratio and assay performance.
- To provide a rapid, homogeneous immunoassay platform for small-molecule contaminant detection.
Main Methods:
- Engineered a homogeneous immunoassay using a split-nanoluciferase (NanoLuc) system, initially as NanoLuc Binary Technology (NanoBiT).
- Developed an enhanced NanoLuc Ternary Technology (NanoTeT) by splitting the NanoLuc 11S subunit into smaller components (Δ11S and β9).
- Labeled anti-parathion nanobody (Nb) VHH9 and artificial antigen H1-ovalbumin (H1-OVA) with specific NanoLuc subunits for assay construction.
Main Results:
- The NanoTeT system achieved a low IC50 of 2.04 ng/mL for parathion, significantly improving sensitivity compared to NanoBiT and ic-ELISA.
- Demonstrated a broad detection range (0.19–22.11 ng/mL) with high accuracy (84.8%–122% recovery) and precision (CV < 15%) in vegetable samples.
- The assay required a simple one-step incubation, completing detection in just 10 minutes, representing a 7-fold increase in speed over ic-ELISA.
Conclusions:
- The developed NanoLuc Ternary Technology (NanoTeT) offers a highly sensitive, rapid, and robust platform for homogeneous immunoassay.
- This novel approach significantly enhances the detection capabilities for small-molecule contaminants like organophosphate pesticides.
- The simplified assay format and improved performance make it suitable for practical applications in food safety and environmental monitoring.
