Date Published: January 27, 2019
Publisher: John Wiley and Sons Inc.
Author(s): Hailong Li, Jesper Vinther Sørensen, Kurt Vesterager Gothelf.
Immunoassays are critical for clinical diagnostics and biomedical research. However, two major challenges remaining in conventional immunoassays are precise quantification and development of immunoassays for small‐molecule detection. Here, a two signal‐mode small‐molecule immunoassay containing an internal reference that provides high stability and reproducibility compared to conventional small‐molecule immunoassays is presented. A system is developed for quantitative monitoring of the digoxin concentration in plasma in the clinically relevant range (0.6–2.6 nm). Furthermore, the model system is integrated into a simple gravity‐driven microfluidic chip (G‐Chip) requiring only 10 µL plasma. The G‐Chip allows fast detection without any complex operation and can be recycled for at least 50 times. The assay, and the G‐Chip in particular, has the potential for further development of point‐of‐care (POC) diagnostics.
Immunoassays have a wide range of applications in clinical diagnostics and molecular biology due to the unique specificity, sensitivity, and flexibility.1 Conventional immunoassays only rely on one‐signal readout channel for (semi)quantification. Moreover, quantitative and specific detection of small molecules remains a great challenge since the conventional sandwich immunoassay, which is capable of a 1000‐fold improvement in detection limit, is not compatible with small‐molecule targets.2 Quantification in particular can be important for small‐molecule drugs that have a narrow therapeutic range, because the assessment of the actual blood concentration in patients can be critical. Furthermore, detection of small molecules with applications in metabolic pathway optimization, metabolite concentration measurement and imaging, environmental toxin detection, and small molecule–triggered therapeutic response is also of interest.3 The most widely used method for measuring concentrations of small molecules is high‐performance liquid chromatography in combination with mass spectrometry (MS), UV, and/or fluorescence detection. These methods are very precise and reliable, but require specialized laboratories and personnel that are trained in the laborious and time‐consuming operation.4
The assay presented here provides a novel method to detect small molecules in plasma using antibodies. It is a type of competitive immunosorption assay, where the presence of the target, here digoxin, binds to the antibody and inhibits further binding. One of the distinct features of the assay is the immobilization of digoxigenin, the aglycon of digoxin, on PS beads linked through BSA. Thus, the excess of the dye‐labeled anti‐Dig is captured on the PS–BSA–Digg beads, whereas the dye‐labeled antibodies that are occupied by digoxin remain in solution after centrifugation or filtration. The fluorescence from the solution is therefore proportional to the concentration of digoxin. One drawback of the method is that it is highly reliable on the quality of the antibody and a certain background from nonbinding antibody is observed in the experiments. The background was constant of ≈40% during experiments. This is probably induced by loss of activity of the antibody in global labeling, and possibly, it may be improved by site‐specific labeling.21, 22
Materials: All chemicals used were of analytical grade and were used without further purification. Functionalized PS beads (Polybead Carboxylate Microspheres 20 µm, 2.5 solids w/v) and PolyLink Protein Coupling Kit were purchased from Polysciences, Inc. (Warrington, PA). Silicon wafers (4 in. were purchased from Corning Inc. (Corning, NY). SU‐8 3050 photoresist and SU‐8 developer were purchased from MicroChem Corp. (Newton, MA). PDMS (RTV615) was purchased from Momentive Performance Materials (Waterford, NY). All devices were designed as computer graphics using AutoCAD software and then printed out as 10 µm resolution film masks by JD Photo Data (Herts, UK). BSA, Atto 680‐labeled streptavidin, polyclonal anti‐Dig antibody from sheep, Atto 488 labeling kit, digoxigenin NHS‐ester, PBS tablet, antibody stabilizer, small molecules, and other associated materials were all purchased from Sigma‐Aldrich. The water used throughout all experiments was purified through a Milli‐Q Biocell System. Organic reactions were monitored by thin‐layer chromatography. Polyacrylamide gels were stained with SimplyBlue SafeStain (Life Technologies) according to manufactures protocol. Antigen–antibody dissociation kit was purchased from LaboratoryEssentials (BioWORLD, Dublin, USA). Refer to the Supporting Information for detailed list of instruments.
The authors declare no conflict of interest.