Immunoassay of Small Molecule Mediated by a Triply Functional DNA

Abstract

Benefiting from specific target recognition by antibodies, the immunoassay is one of the widely used assays for the detection of biologically and environmentally important small molecules in broad fields. It can be challenge to isolate small molecules from their antibody complex in an immobilization-free immunoassay with separation for the detection of small-molecule targets. Here we present an immunoassay mediated by a triply functional DNA probe. A DNA strand is dually labeled with a fluorophore and the target small molecule. This DNA probe integrates three functions, including specific binding to the antibody, signal reporting for sensitive fluorescence detection, and carrying negative charges to facilitate capillary electrophoresis (CE) separation. The binding of the probe to an antibody brings many negative charges in the complex and causes significant changes in mass-to-charge ratios, so the antibody–probe complex can be well separated from the unbound probe in CE analysis. A simple immunoassay is achieved by target competition with this DNA probe for antibody binding in CE coupled to ultrasensitive laser-induced fluorescence (LIF) detection. To show a proof of concept, we detected two model small-molecule targets, digoxin, a therapeutic drug, and ochratoxin A (OTA), an important mycotoxin for food safety. In addition, the use of two DNA probes with distinguished migration times in CE allowed the simultaneous detection of OTA and digoxin. This immunoassay provides new opportunities for wide applications.

Sensitive CRISPR-Cas12a-Assisted Immunoassay for Small Molecule Detection in Homogeneous Solution

“…The sensitivity of this CRISPR-Cas12a-assisted method is better than some of the reported immunoassays with or without signal amplification for digoxin detection. [37][38][39]42,43 The assay was selective for digoxin, and other tested small molecules did not cause interference (Figure S6). Detection of digoxin spiked in a diluted serum was also achieved (Figure S7), and detection limits of digoxin were comparable to those obtained in the assay buffer.…”

Capillary electromigration methods for food analysis and Foodomics: Advances and applications in the period March 2021 to March 2023

“…Two DNA probes were created with specific bindings of the antibody to ochratoxin A and digoxin, with a fluorescence reporter for LIF detection, and carrier negative charges to facilitate the separation of antibody complex using CE. In this manner, ochratoxin A and digoxin were simultaneously separated by CE-LIF in 3 min and detected at nanomolar levels [59].…”

Near-Infrared Light-Initiated Photoelectrochemical Biosensor Based on Upconversion Nanorods for Immobilization-Free miRNA Detection with Double Signal Amplification

“…UCNPs-based PEC biosensors composed of UCNPs and semiconductors with matched energy bands integrate both the merits of UCNPs and PEC assays such as low light injury to biological samples and high sensitivity, , which can be a breakthrough point for molecular signaling detection in complex biological environments. , Simultaneously, the introduction of a nonimmobilization strategy, − i.e., recognition and digestion processes are carried out in solution, can further help to obtain guaranteed sensitivity. , A UCNRs-based PEC sensing platform was developed and realized the sensitive determination of CEA by means of magnetic beads (MB) under NIR light irradiation . After that, a NaYF 4 :Yb,Tm@ZnO upconverter was constructed to detect CEA antigens using a homemade 3D-printed device .…”

“…34,35 Simultaneously, the introduction of a nonimmobilization strategy, 36−38 i.e., recognition and digestion processes are carried out in solution, can further help to obtain guaranteed sensitivity. 39,40 A UCNRs-based PEC sensing platform was developed and realized the sensitive determination of CEA by means of magnetic beads (MB) under NIR light irradiation. 41 After that, a NaYF 4 :Yb,Tm@ZnO upconverter was constructed to detect CEA antigens using a homemade 3D-printed device.…”