Folding Analytical Devices for Electrochemical ELISA in Hydrophobic R Paper

This work describes a device for electrochemical enzyme-linked immunosorbent assay (ELISA) designed for low-resource settings and diagnostics at the point of care. The device is fabricated entirely in hydrophobic paper, produced by silanization of paper with decyl trichlorosilane, and comprises two zones separated by a central crease: an embossed microwell, on the surface of which the antigen or antibody immobilization and recognition events occur, and a detection zone where the electrodes are printed. The two zones are brought in contact by folding the device along this central crease; the analytical signal is recorded from the folded configuration. Two proof-of-concept applications, an electrochemical direct ELISA for the detection of rabbit IgG as a model antigen in buffer and an electrochemical sandwich ELISA for the detection of malarial histidine-rich protein from Plasmodium falciparum (Pf HRP2) in spiked human serum, show the versatility of this device. The limit of detection of the electrochemical sandwich ELISA for the quantification of Pf HRP2 in spiked human serum was 4 ng mL−1 (10 pmol L−1), a value within the range of clinically relevant concentrations. E lectrochemical enzyme-linked immunosorbent assay (ELISA) has the potential to be a useful alternative to conventional colorimetric ELISA for clinical diagnostics because the measured analytical signal is insensitive to color interference and can be converted into a numeric output using inexpensive electronics. Typically, electrochemical ELISA is carried out in standard microtiter plates, with the electroactive product of the enzymatic reaction being transferred at the end to an electrochemical cell to perform the readout. The complexity of the procedure generally limits electrochemical ELISA to well-resourced laboratories with skilled personnel. Easy-to-use, inexpensive, and reliable devices for electrochemical ELISA that can be mass-produced and that are simple enough to be used, with further development, at the point of care, e.g., in the field, in a patient’s home, or in a doctor’s office, would be highly desirable, but they are still not widely available. We wished to develop a device for electrochemical ELISA on paper to provide a foundation for the development of low-cost, fieldable devices for resource-limited settings. Here, we describe the successful fabrication and use of a new, simple family of paper electrochemical devices that can be used for electrochemical ELISA. The devices are made entirely from a sheet of C10 H paper (cellulose paper that has been rendered hydrophobic by vapor-phase silanization with decyl trichlorosilane) and ink, using only embossing, printing, and folding as techniques for fabrication. Despite the remarkable simplicity of these devices (simplicity in the materials used, the fabrication method, and the operation) they are highly sensitive and exhibit the electrochemical behavior typical of conventional electro-