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Fluorescent Fullerene Nanoparticle-Based Lateral Flow Immunochromatographic Assay for Rapid Quantitative Detection of C-Reactive

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Fluorescent Fullerene Nanoparticle-Based Lateral Flow Immunochromatographic Assay for Rapid Quantitative Detection of C-Reactive Park et al. Nano Convergence (2019) 6:35 https://doi.org/10.1186/s40580-019-0207-0 RESEARCH Open Access Fluorescent fullerene nanoparticle-based lateral fow immunochromatographic assay for rapid quantitative detection of C-reactive protein Kyung Mi Park1, Da Jung Chung1, Mijin Choi1, Taejoon Kang2,3* and Jinyoung Jeong3,4* Abstract A fuorescent fullerene nanoparticle (NP)-based lateral fow immunochromatographic assay (LFIA) was developed for the rapid and quantitative detection of C-reactive protein (CRP) in serum. The polyclonal CRP-antibody-conjugated fullerene NPs were simply prepared by 1-ethyl-3-(3-dimethyllaminopropyl)-carbodiimide hydrochloride coupling after carboxylation of fuorescent fullerene NPs. By applying the CRP-antibody-conjugated fullerene NPs to a lateral fow test strip, quantitative analysis of CRP in serum was possible at a concentration range of 0.1–10 ng/ml within 15 min. We anticipate that this novel fuorescent fullerene NP-based LFIA can be useful for the rapid and accurate sensing of biological and chemical species, contributing to the disease diagnosis and prognosis, environmental monitoring, and food safety. Keywords: Fullerene, Nanoparticle, C-reactive protein, Lateral fow immunochromatographic assay, Fluorescence 1 Introduction sensitive and quantitative analyses of molecules even at Te lateral fow immunochromatographic assay (LFIA) low analyte concentrations, challenges remain in terms is a common technique for the detection of such diverse of material preparation, functionalization of the materi- analytes as hormones, disease-related biomarkers, and als for efcient conjugation of the target molecules, and toxins in the clinical, environmental, and food industry optimization of sensing conditions on a lateral fow assay. felds, because of its simplicity and rapidity [1–8]. As a Previously, we developed a novel fuorescent fullerene standard reporting material in LFIA, colloidal gold (CG) material, tetraethylene glycol-conjugated fullerene nano- has been widely used for colorimetric detection due to particles (C60-TEG), that was prepared via a simple pro- its visibility. However, the CG-based LFIA often sufers cedure involving lithium hydroxide as a catalyst at room from limitations such as lack of sensitivity and the abil- temperature [14, 15]. Tese fuorescent fullerene nano- ity to provide only qualitative/semi-quantitative analysis. particles (NPs) are easy to prepare compared to other To overcome the drawbacks of CG-based LFIA, various inorganic materials, i.e., QDs and UCNPs, that require materials have been developed as reporters, including large amounts of surfactants, complex purifcation steps, fuorescent microspheres (FMs), quantum dots (QDs), and harsh conditions such as high temperatures for syn- up-conversion nanoparticles (UCNPs), carbon nano- thesis. Furthermore, the fullerene NPs can provide dis- particles (CNPs), and platinum nanoparticles (PtNPs) tinct and controllable fuorescent signals. Tese unique [9–13]. Although these reporter materials have enabled properties of C60-TEG prompted us to employ them for LFIA. Herein, we report a new fuorescent probe *Correspondence: [email protected]; [email protected] (C -TEG)-based LFIA, for the highly sensitive, rapid, 3 Department of Nanobiotechnology, KRIBB School of Biotechnology, 60 University of Science & Technology, Daejeon 34113, Republic of Korea and quantitative analysis of C-reactive protein (CRP) Full list of author information is available at the end of the article in serum. CRP is known as an acute-phase plasma © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Park et al. Nano Convergence (2019) 6:35 Page 2 of 6 protein that is a non-specifc but sensitive infammation 2 Results and discussion marker, especially in the case of bacterial infection. It 2.1 Synthesis and characterization of pAb‑CRP‑C60‑TEG is also known as a potential indicator of cardiovascular Figure 1 is synthetic procedure of pAb-CRP-C60-TEG for disease, e.g., coronary heart disease, ischemic stroke, LFIA. Firstly, the C60-TEG was prepared by adding LiOH and acute myocardial infarction [16–18]. Because the to a mixture of C60 and TEG. Ten, the NPs were modi- measurement of low concentrations of CRP is critical fed to expose a carboxylate group by the reaction with for early diagnosis of infammation and cardiovascular SA and DMAP. Next, the C 60-TEG-COOH and pAb-CRP disease, many researchers have attempted to develop were conjugated via EDC coupling (Fig. 1). Tis pAb- a highly sensitive CRP-detectable LFIA [19–21]. For CRP-C60-TEG preparation process is uncomplicated example, Swanson et al. recently reported a CRP detec- and easy to perform under ambient conditions, whereas tion limit of 10 ng/ml using near-infrared dye-LFIA other reporting materials, e.g., semiconducting QDs and [22]. In this work, we demonstrated the quantitative UCNPs, are synthesized at high temperatures and in analysis of CRP in the presence of serum with a wide organic solvents. In addition, the C60-TEG-COOH does dynamic range of 0.1–10 ng/ml by using the polyclonal not need to be water-soluble intentionally because it is anti-CRP-conjugated C60-TEG (pAb-CRP-C60-TEG) as highly hydrophilic. a fuorescent probe. Te pAb-CRP-C60-TEG was simply Te absorption and fuorescence spectra of the prepared by 1-ethyl-3-(3-dimethyllaminopropyl)-car- C60-TEG-COOH are shown in Fig. 2a. Te absorption bodiimide hydrochloride (EDC) coupling after carboxy- spectrum shows a strong peak at 272 nm and broad lation of fuorescent fullerene NPs. Since the developed bands at 300–400 nm, indicating the results from car- C60-TEG-based LFIA achieves sufciently high sensitiv- boxylation of C60-TEG which has weak broad band at ity and quantitative analysis of a target molecule, the 260 nm and 350 nm [14]. Tese features are similar to C60-TEG-based LFIA can be used as an advanced fuo- those of the spectra of the hydrophilic fullerene carbox- rescent LFIA for disease diagnosis and prognosis, envi- ylic acid derivative and fullerenol, respectively [23, 24]. In ronmental monitoring, and food safety. addition, the fuorescence spectrum of C60-TEG-COOH exhibits broad fuorescence with a maximum peak at 500 nm under excitation at 350 nm, which is slightly Fig. 1 Schematic illustration of synthetic procedure of pAb-CRP-C60-TEG for LFIA Fig. 2 a Absorption and fuorescence spectra of C60-TEG-COOH. b Infrared spectra of C60-TEG-COOH and pAb-CRP-C60-TEG Park et al. Nano Convergence (2019) 6:35 Page 3 of 6 blue-shifted compared to the fuorescence ofC60-TEG, TL, respectively. After 15 min of migration, the strip was meaning that the carboxylation may lead to change of the subjected to a fuorescence measurement system for the optical properties from C60-TEG. However, the fuores- determination of the TL/CL fuorescence signal ratio. cence quantum efciency of C 60-TEG-COOH (ΦF = 0.33) We optimized the type of blocking solution and its was higher than that of C60-TEG (ΦF = 0.095) so that it is dilution factors to reduce the non-specifc adsorption of sufcient to use as a fuorescent probe for LFIA. pAb-CRP-C60-TEG during fow through the strip. After After conjugating pAb-CRP with C60-TEG-COOH testing three diferent blocking solutions, each contain- via an EDC coupling reaction, we used infrared spec- ing casein, skim milk, and BSA in up to tenfold dilutions, troscopy to examine the antibody conjugation with the we found that 1% BSA solution was the most efcient C60-TEG-COOH. Te infrared spectra in Fig. 2b reveal in obtaining a fuorescence response with a high signal- that the C60-TEG-COOH was successfully conjugated to-noise ratio. Moreover, other parameters, e.g., the vol- with pAb-CRP by the formation of amide bond (C=O ume of sample solution, the washing steps with PBS to amide at 1645 cm−1 and N–H stretch at 3430 cm−1). remove the unbound fuorescent conjugates, and the C–N stretch at 1081 cm−1 shows the presence of amine immunoreaction time, were also optimized to increase bond from the antibody. In comparison, the simple car- the sensitivity. boxylic acid peaks appeared at 1425 cm−1 (OH bend- −1 2.3 Quantitative detection of CRP using C ‑TEG‑based ing) and 1716 cm (C=O stretching) in the spectrum of 60 LFIA unconjugated C60-TEG-COOH. We also determined, via the Bradford assay, that the amount of conjugated pAb- On the basis of the optimal experiment conditions, the CRP was 4.58 µg/mg of C60-TEG-COOH. analytical performance for quantitative measurement of CRP was further evaluated with standard CRP samples in 2.2 Preparation and optimization of C60‑TEG‑based LFIA serum. Figure 4a shows fuorescence images of the TLs By using the pAb-CRP-C60-TEG, we designed the fuo- and CLs on the test strip with various CRP concentra- rescent immunochromatographic assay on a lateral fow tions as high as 10 ng/ml. In the absence of CRP, the CL strip for the rapid and quantitative detection of CRP was easily observed, whereas the TL was not observed. (Fig. 3). First, the pAb-CRP-C60-TEG was dispensed on Both the TL and the CL were visible in the presence of the conjugate pad of a lateral fow test strip. Te anti- CRP, and the lines became more vivid as the CRP con- mouse IgG and mAb-CRP were placed on the nitrocel- centration was increased, indicating that additional pAb- lulose membrane using a dispenser, forming the CL and CRP-C60-TEG was captured on the TL.
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