Inner Filter Effect-Based Sensor for Horseradish Peroxidase and Its Application to Fluorescence Immunoassay

Abstract

Being an important model peroxidase, horseradish peroxidase (HRP) has been thoroughly understood, and the detection of HRP is not only directly related to peroxidasetriggered catalytic process, but also linked to the development of HRP-based enzyme-linked immunosorbent assay (ELISA). Herein, we have reported an unconventional fluorescent sensor for convenient assay of HRP activity based on the HRPcatalyzed specific conversion of p-phenylenediamine (PPD) into chromogenic PPDox with H 2 O 2 as the oxidizing agent, accompanied by the fluorescence quenching effect on fluorescein. By combining UV−vis absorption spectrum, isothermal titration calorimetry, and fluorescence lifetime analysis, we have confirmed the inner filter effect as a main quenching mechanism in our proposed fluorescent assay. According to the intrinsic sensitivity of fluorescent sensor and high selectivity, our PPD/fluorescein-based sensing system can be utilized for real-time monitoring of the HRP activity in real biological samples. Furthermore, the unambiguous response mechanism and excellent sensing performance encourage us to extend such HRP assay into the HRP-based fluorescent ELISA, which has a broad prospect of application in fluorescent diagnosis of hepatocellular carcinoma (HCC) by sensing alphafetoprotein, the well-known serologic HCC marker.

A Janus-inspired amphichromatic system that kills two birds with one stone for operating a “DNA Janus Logic Pair” (DJLP) library

“…S5b †), thus eliminating the static quenching that was caused by the production of a non-uorescent ground-state complex. 60,61 Thus, we speculated that the luminescence quenching between UCP and OxTMB could be attributed to the inner lter effect, which was also consistent with other recent reports. 60,61 To dene the amphichromatic outputs more accurately, normalized Abs (650 nm) or UCL (545 nm) was used to discriminate the high/low outputs and 0.45 was taken as the universal threshold ( Fig.…”

“…For the quenching mechanism, the following observations were made: (1) the lifetimes (Fig. 1g) of UCP before (green line, 115.2 ms) and aer adding OxTMB (blue line) were almost unchanged, proving that dynamic quenching was not the main reason; 60,61 (2) aer mixing with UCP, OxTMB's absorbance changed slightly (Fig. S5a †) and the reaction product that was catalyzed by non-G4 DNAs/hemin could not quench the UC emission ( Fig.…”

A Ratiometric Fluorescent DNA Radar Based on Contrary Response of DNA/Silver Nanoclusters and G-Quadruplex/Crystal Violet

“…The fluorescence lifetime shortened obviously in the presence of CV, excluding the inner filter effect of fluorescence (Figure S5D). 48,49 (v) HR-TEM results demonstrated that CV with increased concentration could induce the growth of size and aggregation of AgNCs (Figure S6), which might be the change of the AgNC luminescence center. 50 (vi) The fluorescence intensity of Probe-22 bp with prepared AgNCs at 625 nm was stronger than that of the probe without prepared AgNCs (Figure S7) when putting in the same amount of Com, which meant that the new emission might not only be derived from Com but also a fraction from the FRET effect.…”

Fluorescent Carbon Dots as Biosensor, Green Reductant, and Biomarker

“…Fluorescence quenching may occur via various ways such as (i) static quenching and ground-state complex formation between the fluorophore and the analyte, (ii) dynamic quenching, (iii) Forster resonant energy transfer (FRET), (iv) photoinduced electron transfer (PET), the inner filter effect (IFE), and so forth. 65 Therefore, it is imperative to figure out the exact mechanism that leads to the quenching of fluorescence of the M-CDs to remove the ambiguity regarding the selectivity. An analysis to this effect rules out the chance for FRET as there is no overlap between the absorption spectrum of FA and the emission spectrum of M-CDs ( Figure 5 b).…”