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Enzyme-linked immunosorbent assays (ELISA), as one of the most used immunoassays, have been conducted ubiquitously in hospitals, research laboratories, etc. However, the conventional ELISA procedure is usually laborious, occupies bulky instruments, consumes lengthy operation time, and relies considerably on the skills of technicians, and such limitations call for innovations to develop a fully automated ELISA platform. In this paper, we have presented a system incorporating a robotic-microfluidic interface (RoMI) and a modular hybrid microfluidic chip that embeds a highly sensitive nanofibrous membrane, referred to as Robotic ELISA, to achieve human-free sample-to-answer ELISA tests in a fully programmable and automated manner. It carries out multiple bioanalytical procedures to replace the manual steps involved in classic ELISA operations, including the pneumatically driven high-precision pipetting, efficient mixing and enrichment enabled by back-and-forth flows, washing, as well as integrated machine vision for colorimetric readout. The Robotic ELISA platform has achieved a low limit of detection (LOD) of 0.1 ng/mL in the detection of a low sample volume (15 μL) of chloramphenicol (CAP) within 20 min without human intervention, which is significantly faster than that of the conventional ELISA procedure. Benefiting from its modular design and automated operations, the Robotic ELISA platform has great potential to be deployed for a broad range of detections in various resource-limited settings or high-risk environments, where human involvement needs to be minimized, while the testing timeliness, consistency and sensitivity are all desired.

An ultrasensitive and portable colorimetric enzyme-linked immunosorbent assay (ELISA) sensor for antibiotics was fabricated by immobilizing antibodies inside the largely porous and highly hydrophilic nanofibrous membranes. Different from regular electrospun nanofibrous membranes where antibodies may frequently be blocked by the heterogeneous porous structure and sterically crowded loaded on the surface, the controlled microporous structure and increased hydrophilicity of nanofibrous membranes could improve the diffusion properties of antibodies, reduce the sterically crowding effect, and dramatically improve the sensitivity of the membrane-based ELISA. The limitation of detection (LOD) for chloramphenicol (CAP) reached 0.005 ng/mL, around 200 times lower than the conventional paper-based ELISA, making quantitative analysis and portable on-site detection achievable via the use of smartphones. The successful design and fabrication of the nanofibrous membrane-based ELISA with novel features overcome the structural drawbacks of regular electrospun nanofibrous membranes and provide new paths to develop highly sensitive on-site detection of hazardous chemical agents.

Enzyme-linked immunoassay (ELISA) is highly specific and selective towards target molecules and is convenient for on-site detection. However, in many cases, lack of high sensitivity makes it hard to reveal a significant colorimetric signal for detecting a trace amount of target molecules. Thus, analytical instruments are required for detection, which limits the application of ELISA for on-site detection. In the present study, a highly sensitive and naked-eyed detectable colorimetric biosensor for chloramphenicol (CAP) was prepared by incorporating ELISA onto surfaces of microporous and nanofibrous membranes. The high specific surface areas of the nanofibers significantly increased the number of antibodies covalently linked onto the fiber surfaces and binding capacity of the sensor with antigens present in a sample. With such an integration, the sensitivity of the ELISA sensor was dramatically increased, and a trace number of targets could reveal a naked-eye detectable color. The immunoassay sensor exhibited a significant naked-eye distinguishable color to chloramphenicol (CAP) at 0.3ng/mL. The successful design and fabrication of the nanofibrous membrane immunoassay sensor provide new paths towards the development of on-site inspection sensors without the assistance from any instrument.