Synthesis and grafting of diazonium tosylates for thermoplastic electrode immunosensors

Abstract

For electrochemical immunosensors, inexpensive electrodes with fast redox kinetics, and simple stable methods of electrode functionalization are vital. However, many inexpensive and easy to fabricate electrodes suffer from poor redox kinetics, and functionalization can often be difficult and/or unstable. Diazonium tosylates are particularly stable soluble salts that can be useful for electrode functionalization. Recently developed thermoplastic electrodes (TPEs) have been inexpensive, moldable, and highly electroactive carbon composite materials. Herein, the synthesis and grafting of diazonium tosylate salts were optimized for modification of TPEs and used to develop the first TPE immunosensors. With diazonium tosylates, TPEs were amine functionalized either directly through grafting of p-aminophenyl diazonium salt or indirectly through grafting p-nitrophenyl diazonium salt followed by electrochemical reduction to an amine. Diazonium tosylates were synthesized in situ as a paste in 6 min. Once the reaction paste was spread over the electrodes, near monolayer coverage (1.0 ± 0.2 nmol cm^-2) was achieved for p-nitrophenyl diazonium salt within 5 min. Amine functionalized electrodes were conjugated to C-reactive protein (CRP) antibodies. Antibody-modified TPEs were applied for the sensitive detection of CRP, a biomarker of cardiovascular disease using electrochemical enzyme-linked immunosorbent assays (ELISA). LODs were determined to be 2 ng mL^-1 in buffer, with high selectivity against interfering species for both functionalization methods. The direct p-aminophenyl modification method had the highest sensitivity to CRP and was further tested in spiked serum with an LOD of 10 ng mL^-1. This low-cost and robust TPE immunosensor platform can be easily adapted for other analytes and multiplexed detection.

Fig. 1.

P-nitroaniline modification optimization and stability testing. A) Pretreatment optimization (n=4), B) sonication time in IPA following modification (n=4), C) modification time and conditions: open air (5 and 10 min n=5; 15 min n=7) and humidified petri dish (5 min n=9; 10 and 15 min n=5), and D) Coverage stability.

Fig. 2.

Averaged cyclic voltammetry of modified 1 mm diameter TPEs in N₂ degassed 0.1 M H₂SO₄ v=100 mV/s, modified with and without NaNO₂. A) p-nitrophenyl diazonium salt modification (with NaNO₂ n=16; without NaNO₂ n=6), B) p-aminophenyl diazonium salt modification (with NaNO₂ n=6; without NaNO₂ n=6).

Fig. 3.

Streptavidin-ALP assay data for reduced nitrophenyl, direct aminophenyl, and reduced aminophenyl modified electrodes from ALP generated p-AP SWV. A) Quantitative blocking optimization peak current data (BSA n=6; SB and BSA+SB n=3), B) averaged voltammograms for biotinylated TPEs and unbiotinylated controls (biotinylated n=6; controls n=3), C) quantitative peak area data for biotinylated electrodes, and D) quantitative peak area data for unbiotinylated controls.

Fig. 4.

CRP ELISA assay p-AP peak area data for reduced nitrophenyl (Nitrophenyl) modified electrodes and reduced aminophenyl (Aminophenyl) modified electrodes for various concentration of CRP in PBS buffer pH=7.4 (0.1-10⁴ ng/mL; n=4 for all but PPD 10³ ng/mL [n=3]), CRP spiked 100x diluted serum (1-10³ ng/mL; n=3), and 10³ ng/mL myoglobin (n=3).

Scheme 1.

Diazonium salt synthesis and modification schematics. A) p-nitrophenyl diazonium salt modification and subsequent reduction of nitro groups to amines, B) p-aminophenyl diazonium salt modification, and C) reaction and modification images with p-nitrophenyl diazonium salt shown.

Scheme 2.

Assay modification schematics. A) Streptavidin-ALP biotin proof of concept assay. B) CRP ELISA assay.