Elsevier

Biosensors and Bioelectronics

Volume 150, 15 February 2020, 111885
Biosensors and Bioelectronics

Gold nanocap-supported upconversion nanoparticles for fabrication of a solid-phase aptasensor to detect ochratoxin A

https://doi.org/10.1016/j.bios.2019.111885Get rights and content

Highlights

  • Concept & design of solid-phase, single-step biosensors were demonstrated.

  • Fabrication based on Au nanocap-supported upconversion nanoparticles on PDMS.

  • Quencher-labeled aptamers functionalized onto exposed Au/nanoparticle area.

  • Sensitivity toward ochratoxin A was comparable to that of solution-phase sensor.

  • Reusability of solid-phase single-step aptasensor was successful via heat treatment.

Abstract

Solid-phase, single-step biosensors are crucial for the development of portable, reusable, and convenient biosensors, otherwise known as point-of-care (POC) testing. Although high-performance single-step biosensors based on the principle of Förster resonance energy transfer (FRET) and using upconversion nanoparticles (UCNPs) functionalized with aptamers have been suggested as easy-to-use platforms, they lack portability and reusability when used for solution-phase biosensing. In this study, we describe a solid-phase, single-step aptasensor that showed higher performance than those of solution-phase aptasensors, as well as promising reusability. The solid-phase, single-step aptasensor was developed based on Au nanocap-supported UCNPs (Au/UCNPs), which were partially embedded in a solid substrate (e.g. polydimethylsiloxane, PDMS). The Au nanocaps allowed the UCNPs to emit upconverted light only from the restricted areas of the UCNPs, i.e., where they were not covered by the nanocaps and PDMS. Functionalization of an aptamer labeled with a quencher on the restricted area enabled the effective quenching of upconverted light from Au/UCNP via FRET after target (ochratoxin A, OTA) detection. The solid-phase, single-step aptasensor showed a linear range of 0.1–1000 ng mL−1 and limit of detection of 0.022 ng mL−1 within 30 min toward OTA. Furthermore, reusability of the solid-phase aptasensor was evaluated for three cycles of detection and regeneration, establishing its apparent reusability via heat treatment. Hence, such solid-phase, single-step aptasensors pave the path to the development of a portable and reusable biosensor platform for POC testing.

Introduction

Portable and easy-to-use biosensors are highly important for future healthcare systems that are individual-centered, also known as point-of-care (POC), rather than laboratory- or hospital-centered (Gubala et al., 2012; Vashist et al., 2015; Nayak et al., 2017; Wood et al., 2019). Based on rapid advances in nanomaterial-based technology, various designs for nanoparticle–bioreceptor conjugates dispersed in solution have led to the development of sensitive biosensor platforms that are potentially applicable for POC (Luppa et al., 2011; Quesada-González and Merkoçi, 2018; Samorì and Biscarini, 2018; Ye et al., 2018). In particular, upconversion nanoparticles (UCNPs) have attracted much attention as strong candidates for such conjugates because they can be excited by near-infrared light without photodamage or matrix effects and emit highly stable visible light (Zheng et al., 2015; Sedlmeier and Gorris, 2015; Gu and Zhang, 2018; Ye et al., 2014; Xu et al., 2014; Jo et al., 2018). To take advantage of these properties, the functionalization of bioreceptors on UCNPs has been explored to develop target-specific and high-performance biosensor platforms based on quenching of the upconversion luminescence (UCL) of UCNPs in the presence of target molecules via Förster resonance energy transfer (FRET) (Ye et al., 2014; Xu et al., 2014; Jo et al., 2018). Despite the high sensitivities achieved by nanoparticle–bioreceptor conjugates dispersed in solution, the development of biosensors for POC testing remains a challenge due to their poor portability, the aggregation of the conjugates, reusability issues, and their complexity of use.

Solid-phase, single-step biosensors are regarded as strong candidates to solve such challenges, as the immobilization of nanoparticles, bioreceptors, and their conjugates on solid substrates provides a highly portable reusable platform, and avoids the issue of nanoparticle aggregation (Guan et al., 2015; Han et al., 2017). In addition, single-step biosensors are facile to employ, as no centrifuge equipment is required for the washing steps, and the process tends to be less time-consuming (Jo et al., 2018). In the context of single-step biosensors, our group has recently reported a conjugate (i.e., a UCNP functionalized with quencher (organic dye)-labeled aptamers) that enables highly sensitive single-step detection of small molecules, and is a highly convenient biosensor platform for individual users because of its single-step procedure for target detection (Jo et al., 2018). Nevertheless, such biosensors based on conjugates dispersed in solution (also known as solution-phase biosensors) have considerable limitations in terms of the development of POC biosensors as they do not take advantage of solid-phase biosensors.

Although there have been a few studies on the immobilization of UCNPs on solid substrates and subsequent functionalization of bioreceptors onto the UCNPs to develop platforms for solid-phase biosensors (Doughan et al., 2014, 2015), solid-phase, single-step biosensors with sensitivities comparable to solution-phase ones have not yet been reported. Therefore, biosensors satisfying solid-phase and single-step are highly demanded for the development of future POC biosensors.

In this study, we demonstrated solid-phase, single-step biosensors based on Au nanocap-supported UCNPs (Au/UCNPs) partially embedded in a solid substrate (e.g., polydimethylsiloxane, PDMS), where quencher-labeled aptamers were functionalized on the UCNPs to detect ochratoxin A (OTA). The Au/UCNPs can emit strong and directional upconverted light from only the restricted surface of the Au/UCNPs that is not covered by the Au nanocap (Scheme 1); this strong and directional upconversion emission from the restricted area of the UCNPs stems from plasmon enhancement of the UCL caused by the Au nanocap (Bang et al., 2017; Hinamoto et al., 2017). Then, quencher-labeled aptamers were covalently functionalized on the restricted surface of Au/UCNPs, where the UCNPs were not covered by the Au nanocap. Thus, the quencher-labeled aptamers could effectively quench the directionally emitted light from the Au/UCNPs via FRET depending on the OTA concentration, which showed comparable and even higher sensitivity to that of solution-phase biosensors. The higher sensitivity is expected possibly because of enhanced FRET efficiency originating from increased UCL of UCNPs and/or plasmon-assisted FRET by Au nanocap. Finally, the solid-phase, single-step aptasensors were successfully applied as reusable biosensors via heat treatment. Hence, the fabrication of aptamer-functionalized Au/UCNPs on PDMS could pave the way for the development of solid-phase, single-step biosensors.

Section snippets

Materials

Yttrium(III) acetate hydrate (Y(CH3COO)3·xH2O, 99.9% metals basis), ytterbium(III) acetate hydrate (Yb(CH3COO)3·xH2O, 99.95% trace metals basis), erbium(III) acetate hydrate (Er(CH3COO)3·xH2O, 99.9% trace metals basis), oleic acid (technical grade, 90%), 1-octadecene (technical grade, 90%), ammonium fluoride (NH4F, ACS reagent, ≥98.0%), sodium hydroxide (NaOH, BioXtra, ≥98% (acidimetric), pellets (anhydrous)), methanol (for HPLC, ≥99.9%), cyclohexane (ACS reagent, ≥99%), OTA, ochratoxin B

Results and discussion

The fabrication of aptamer-functionalized Au/UCNPs on PDMS as a solid-phase, single-step biosensor is described in Fig. 1a and b. In brief, a solution containing UCNPs (characterized in detail in Fig. S1) was drop-cast onto a glass substrate and dried (Fig. 1a). Scanning electron microscopy (SEM) imaging of the resulting UCNP-coated glass substrate demonstrated the formation of a UCNP monolayer (Figs. S2a and b). Then, Au was deposited using an e-beam evaporator to form Au nanocaps on UCNPs (

Conclusions

In this paper, we demonstrated a solid-phase, single-step aptasensor based on Au nanocap-supported UCNPs functionalized with quencher-labeled aptamers on a PDMS substrate. The Au/UCNP-based solid-phase aptasensor was used to detect OTA within the range of 0.1–1000 ng mL−1 on the basis of quenching of the UCL of UCNPs caused by FRET between the UCNPs and quencher. The role of the Au nanocaps on the UCNPs was to restrict the strong UCNP emission of upconverted light to a specified area due to

CRediT authorship contribution statement

Kihyeun Kim: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review & editing. Eun-Jung Jo: Methodology, Formal analysis, Investigation, Writing - original draft. Ki joong Lee: Methodology, Formal analysis, Investigation. Jiyoon Park: Methodology, Investigation. Gun Young Jung: Investigation, Writing - review & editing. Yong-Beom Shin: Formal analysis, Investigation, Writing - review & editing. Luke P. Lee:

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgment

This work was financially supported by grants from the Global Research Laboratory (GRL) Program (NRF-2013K1A1A2A02050616); the Mid-Career Researcher Program (NRF-2017R1A2B3010816) through a National Research Foundation grant funded by the Ministry of Science, ICT, and Future Planning; BioNano Health-Guard Research Center funded by the Ministry of Science and ICT (MSIT) of Korea as a Global Frontier Project (Grant number H-GUARD_2013M3A6B2078950); and GIST Research Institute (GRI) grant funded

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