Ratiometric electrochemical proximity assay for sensitive one-step protein detection

Abstract

This work proposes the concept of ratiometric electrochemical proximity assay (REPA), which can be used for one-step, highly sensitive and selective detection of protein. The assay strategy was achieved on a sensing interface that was formed by hybridization of methylene blue (MB)-labeled antibody-DNA probe (MB-DNA1-Ab1) with ferrocene (Fc)-labeled DNA capture probe (Fc-P) modified gold electrode. On the interface the target protein could trigger the formation of immunocomplex between MB-DNA1-Ab1 and detection antibody-DNA probe (Ab2-DNA2) and subsequently the proximity hybridization of DNA1-DNA2, which led to the departure of MB-DNA1-Ab1 from the interface. The remained Fc-P could form a hairpin structure to take Fc group to electrode surface. Therefore, the recognition of target protein to Ab1 and Ab2 resulted in both the “signal-off” of MB and the “signal-on” of Fc for dual-signal electrochemical ratiometric readout. The proposed REPA could be carried out in one-step with 40-min duration and showed a wide detection range from 0.05 to 100 ng/mL with pg/mL limit of detection, displaying great potential for convenient point-of-care testing and commercial application.

A new ratiometric electrochemical sensor for sensitive detection of bisphenol A based on poly-β-cyclodextrin/electroreduced graphene modified glassy carbon electrode

“…The detection limit of the proposed sensor is also lower than those obtained by other electrochemical methods reported previously [1,[11][12][13][14][15][16][17][18][19][20][21][22][23] (Table 1). The predominant reasons might be as follows: (1) the good characteristics of the ratiometric electrochemical assay (self-calibration, low detection limit) [43,[60][61]; (2) the synergistic effect of the electro-reduced graphene (high surface area and good electron transfer property) and Pβ-CD (lots of CD units, the excellent enrichment and host-guest recognition properties of CD). thin-film electrode array 44-4400 44 [11] Graphene/GC 80-5000 20 [12] CTAB/GC 600-100000 100 [13] peptide/Au electrode 1-5000 0.7 [14] SWCNT-CD/GC 10.8-18500 1 [15] CS/N-GS/GC 10-1300 5 [16] Gr-SP-Tyr/GC 2-5480 0.72 [17] Laccase-thionine-CB/SPCE 500-50000 200 [18] AuNPs/SGNF/GC 80-250000 35 [19] SWCNTs-COO-Poly-IL/GC 5-30000 1 [20] PGA/MWCNT-NH2/GC 100-10000 20 [21] MWCNTs/CoPc/SF/GC 50-3000 30 [22] Tyr-NiNPs/SPCE 910-48000 7.1 [23] RhB-incubated Similarly, no interference is observed for 1-naphthols, 2-naphthols, 4-aminophenol (5 equiv).…”

“…The developed modified electrode (Pβ-CD/EG/GC) was successfully used to detect BPA, based on the enrichment function of CDs and electrochemical oxidation signal of BPA. In order to enhance the sensitivity of the developed sensor, ratiometric analysis strategy was adopted due to their good characteristics (such as self-calibration, low detection limit) [40][41][42][43] and Rhodamine B (RhB) was used as the probe (Scheme 1). The resulted BPA sensor shows wide linear range, low detection limit, excellent selectivity, as well as good reproducibility.…”

A ratiometric electrochemical aptasensor for sensitive detection of protein based on aptamer–target–aptamer sandwich structure

“…As a result, the MB tag is pushed farther away from the electrode surface and the two Fc tags are dragged closer to the electrode surface compared with those before addition of thrombin, leading to a decrease of the MB signal (I MB ) and an increase of the Fc signal (I Fc ). Detection of thrombin can be realized by simultaneously monitoring the ratio (I Fc /I MB ) between I Fc and I MB due to the good characteristics of ratiometric detection (such as self-calibration, low detection limit) [32,37]. The resulted E-AB sensor shows wide linear range, low detection limit, excellent selectivity, as well as good reproducibility.…”

“…However, many targets, such as thrombin [28,29], platelet-derived growth factor [30,31], prostate specific antigen [32], and vascular endothelial growth factors [33] and MUC1 protein [34], have more than one sites to bind their aptamers or antibodies to form a sandwich structure, which has an obviously better anti-interference ability than the simple biosensing structure [35]. Although lots of E-AB biosensors have been developed to detect these targets based on single-signaling strategy, so far no dual-signaling E-AB biosensors have utilized the aptamer-target-aptamer sandwich structure with both aptamers labeled by redox species for robust detection of the target.…”

Signal Amplification for Highly Sensitive Immunosensing

“…For example, Landegren et al [83] combined proximity ligation assay (PLA) using DNA-bound antibodies to recognize the target molecule with RCA amplification to develop a method for highly sensitive protein detection. Ju et al [84] designed a ratiometric electrochemical proximity assay for one-step sensitive detection of protein by combination of the ratiometric detection technology with the proximity hybridization. As shown in Fig.…”