Efficient Optosensing of Hippuric Acid in the Undiluted Human Urine with Hydrophilic "Turn-On"-Type Fluorescent Hollow Molecularly Imprinted Polymer Microparticles

Abstract

The development of complex biological sample-compatible fluorescent molecularly imprinted polymers (MIPs) with improved performances is highly important for their real-world bioanalytical and biomedical applications. Herein, we report on the first hydrophilic "turn-on"-type fluorescent hollow MIP microparticles capable of directly, highly selectively, and rapidly optosensing hippuric acid (HA) in the undiluted human urine samples. These fluorescent hollow MIP microparticles were readily obtained through first the synthesis of core-shell-corona-structured nitrobenzoxadiazole (NBD)-labeled hydrophilic fluorescent MIP microspheres by performing one-pot surface-initiated atom transfer radical polymerization on the preformed "living" silica particles and subsequent removal of their silica core via hydrofluoric acid etching. They showed "turn-on" fluorescence and high optosensing selectivity and sensitivity toward HA in the artificial urine (the limit of detection = 0.097 μM) as well as outstanding photostability and reusability. Particularly, they exhibited much more stable aqueous dispersion ability, significantly faster optosensing kinetics, and higher optosensing sensitivity than their solid counterparts. They were also directly used for quantifying HA in the undiluted human urine with good recoveries (96.0%-102.0%) and high accuracy (RSD ≤ 4.0%), even in the presence of several analogues of HA. Such fluorescent hollow MIP microparticles hold much promise for rapid and accurate HA detection in the clinical diagnostic field.

Keywords: complex biological samples; fluorescence “turn-on”; hippuric acid; hollow; human urine sample; molecularly imprinted polymers; sacrificial template method; surface-initiated ATRP.

Scheme 1

(a) Schematic illustration for preparing “turn-on”-type fluorescent hollow MIP microparticles with PEG brushes via the combined use of one-pot SI-ATRP strategy and the sacrificial template method. (b) Chemical structures of some reagents utilized in this work.

Figure 1

(a) AFM height images of SiO₂-Br (a), SiO₂@NBD-MIP@PEG (b), SiO₂@NBD-CP@PEG (c), H@NBD-MIP@PEG (d), and H@NBD-CP@PEG (e). The scale bar is 1 μm.

Figure 2

(a) FT-IR spectra of SiO₂-Br (a1), SiO₂@NBD-MIP@PEG (a2), SiO₂@NBD-CP@PEG (a3), H@NBD-MIP@PEG (a4), and H@NBD-CP@PEG (a5). (b) Photographs of the ultrasonically dispersed aqueous mixtures (2.0 mg/mL; the concentration of the hollow samples was calculated by using the weights of their solid counterparts before etching) after being settled down at 20 °C for 0 h (b1,b2), 20 h (b3,b4), and 34 h (b5,b6), respectively. The samples located from left to right in each photograph are pure water, SiO₂-Br, SiO₂@NBD-MIP@PEG, SiO₂@NBD-CP@PEG, H@NBD-MIP@PEG, and H@NBD-CP@PEG [the photographs of the aqueous mixtures were taken under the irradiation of the natural light (b1,b3,b5) and 365 nm UV light (b2,b4,b6)].

Figure 3

(a,b) Fluorescence spectra of the hydrophilic fluorescent hollow HA-MIP (a)/CP (b) after their incubation with a HA solution (20 μM) in the artificial urine at 25 °C for different times (hollow MIP/CP concentration: 0.25 mg/mL; their concentration was calculated by using the weight of their corresponding solid ones before etching). (c) Optosensing kinetics of the hydrophilic fluorescent hollow HA-MIP (filled symbol)/CP (open symbol) in a HA solution (20 μM) in the artificial urine at 25 °C (derived from Figure 3a,b; F_t and F₀ in (F_t − F₀)/F₀ are the fluorescence intensity of the NBD unit (at 514 nm) at a time of t and 0, respectively).

Figure 4

(a,b) Fluorescence spectra of the hydrophilic fluorescent hollow HA-MIP (a)/CP (b) upon their exposure to different concentrations of HA in the artificial urine at 25 °C for 2 h (hollow MIP/CP concentration: 0.25 mg/mL; their concentration was calculated by using the weights of their solid counterparts before etching). (c) Dependence of the fluorescence enhancement [(F − F₀)/F₀, where F and F₀ are the fluorescence intensity of NBD fluorophores at 514 nm in the presence and absence of HA, respectively] of the hydrophilic fluorescent hollow HA-MIP (filled symbol)/CP (open symbol) on the HA concentration (derived from Figure 4a,b).

Figure 5

Fluorescence enhancement of the hydrophilic fluorescent hollow HA-MIP (filled column)/CP (open column) upon exposure to a HA, 3-MHA, 4-AHA, or Tyr solution (C_{HA, 3-MHA, 4-AHA, or Tyr} = 20 μM) (a) or to a HA solution (20 μM) in the presence of 40 μM of 3-MHA, 4-AHA, or Tyr (b) in the artificial urine at 25 °C for 2 h (hollow MIP/CP concentration: 0.25 mg/mL; their concentration was calculated by using the weights of their solid counterparts before etching).