Elsevier

Analytical Biochemistry

Volume 556, 1 September 2018, Pages 129-135
Analytical Biochemistry

Development of a lateral flow immunoassay of C-reactive protein detection based on red fluorescent nanoparticles

https://doi.org/10.1016/j.ab.2018.06.017Get rights and content

Highlights

  • A facile one-step synthesis is used to prepare single microspheres for CRP assay.

  • Clear core-shell structures of microspheres contribute to divide functional areas.

  • Separating antibody and dyes to improve the sensitivity and stability of test strip.

  • An analysis of clinical samples is used to get the reliable assay results.

Abstract

We have developed a reliable and rapid immunoassay based on a facile synthesis of fluorescent nanoparticles integrated in immunochromatography technique to quantitatively detect C-reactive protein (CRP). The method is based on a sandwich immunoassay using the Nile-red doped nanoparticles/CRP monoclonal antibody conjugate. The method is simple and fast, with a detection limit of 0.091 mg/L. It provides quantitative analysis in the range of 0.1–160 mg/L, which is adequate for detecting CRP of acute inflammatory or cardiovascular disease. This strategy displayed a good reproducibility and stability to straightforwardly analyze the plasma samples without complicated washing steps, thereby reducing the operating procedures for non-professionals and promoting the detection efficiency and the whole detection process can be completed in 3 min. This approach for carrying out immunoassays can be applied to the detection of CRP in the point-of-care tests.

Graphical abstract

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Schematic presentation of lateral flow immunoassays (LFIA) based on the use of red fluorescent nanoparticles (RPNs) as signaling labels for the rapid determination of C-reactive protein (CRP) in plasma samples and the core-shell structure of the RPNs.

Introduction

The C-reactive protein (CRP) is first discovered in the serum of the infected patients in 1930 and is well known as an acute phase biomarker of the immune response in humans nowadays. The concentration of CRP is routinely determined in blood counts and other clinical diagnostics. In normal blood serum, CRP is range from 1 to 5 mg/L [1]. Its concentration can be increased up to 1000 folds [2] in serum after acute inflammatory events such as tissue injury, trauma, and surgery. Also, the association between high sensitivity CRP (hs-CRP) and cardiovascular diseases have been well studied, especially the coronary vascular disease (CVD). Patients that detected a higher hs-CRP values with stable angina pectoris may have a greater CVD risk, or even among the health subjects that have a higher risk of CVD when the hs-CRP value is over 3 mg/L [3].

The clinical analysis of routine CRP-check in patients are measured by enzyme linked immune sorbent assay (ELISA) kits with a limit of detection down to 0.2 mg/L [4], or the testing method based on turbidimetric and nephelometric immunoassay technologies [5]. ELISA assay is accurate for the test of the CRP levels, however, it is generally not suitable for the point-of-care tests (POCT) applications since the analytical process is time-consuming, normally required about 2 h. The immunoturbidimetry is reliable, while the stability period of the kits is general about one month, thus could not meet the actual application that may need much more additional time.

In recent years, there are many developments for the measurement of CRP. CRP quantification based on immune precipitation have been reported with a detection limit of 25 ng/μL [6]. Another method to detect the CRP levels has been carried out by utilizing the bead-based aptamer/antibody detection system within a detection limit of 0.4 mg/L [7]. Higher sensitive method was developed based on the optical fiber Bragg within the antibody-graphene oxide complex. The detection limit of this assay was 0.01 mg/L [8], and the RNA aptamer-based capacitive label-free biosensor for the detection of CRP was reported with higher sensitive [9]. Alternatively, a label-free biosensor based on reflectometric interference spectroscopy technology was developed to measure the CRP levels [10]. The above mentioned approaches to detect CRP with higher sensitivity and lower limit of detection, while the clinical application of these assays were limited by the demand for costly device, skilled staff and cumbersome operation.

Therefore, developing an accurate, more rapid, portable, and user-friendly analytical technique which referred as POCT is of great important in clinical field. Compared to central laboratory, POCT provides timely and accurate results, which the analysis can finish in 30 min [11]. Lateral flow immunoassays (LFIA) also known as immunochromatographic assays is a popular POCT diagnostic mean since its advantages of quick, one-step, in-situ analysis, and easy to use. The conventional LFIA used gold nanoparticles (AuNP) as reporters, most of them can only have the qualitative results, also there were AuNP based LFIA can offer the quantitative data, that commonly used to analyze medium or high concentrations of analytes [12]. Therefore, the development of a high sensitivity of LFIA which can offer quantitative data even at low sample levels and in complex matrices has been constantly increased over past decades based on the fluorescent immunoassay. However, most of the traditional fluorescent probes present a drawback that the emission is located in the area which include the autofluorescence of the samples, resulted in unsatisfied sensitivity [13]. Therefore, people developed a range of probes such as organic dyes, up conversion rare-earth materials and quantum dots to improve the sensitivity and performance of the LFIA in recent years [14,15]. Most of them showed a high sensitivity, but displayed a complicated method and costly materials in the synthesis of the fluorescent probes.

In this work, we exhibited a simple and cost effective method to produce a dye-doped fluorescent probe with high quality and accompany with satisfied performance of the LFIA in the immunoassay of CRP. Generally, the organic dye is combined directly with antibody or biotin in the immunoassay [16], which are usually exposed to the unsatisfied environmental conditions, suffering from photobleaching and quenching [17]. In order to improve the performance of the organic dye, the dye-doped nanoparticles were developed and instead of the dye molecules in recent years. The dye labeled nanoparticles with higher brightness and stability compared with the dye molecules, resulted in higher reproducibility in practical application. Furthermore, in the aim of achieving the coupling reaction between the dye-labeled-nanoparticles and the antibody, the nanoparticles with functional groups to capture the antibody were also synthesized in this study. Among the various material nanoparticles, such as silica nanoparticles, graphene-metal oxide composite nanoparticles, and Fe3O4 modified nanoparticles [[18], [19], [20]], the polystyrene based nanoparticles as common material have obvious advantages, such as easily controlling the functional groups, simple synthetic steps, high yield and low cost, which promote their application in practical used.

In this paper, a dye-labeled polystyrene nanoparticle was synthesized to establish a new POCT method for the rapid and broad detection range of CRP diagnosis. The fluorescent nanoparticle used Nile-red (NR) as the fluorescent dye within 608 nm emission wavelength, which efficiently deceased the interference signal of the background noise. Besides, since the developed technique has also improved the processing step with one step sampling and without any washing step, is rapid and friendliness to the non-professionals.

Section snippets

Materials and reagents

All chemicals and solvents were of analytical grade. The potassium peroxodisulfate (KPS), styrene (98%) and dichloromethane were purchased from Aladdin Biochemical Technology Co., Ltd. (Shanghai, China, www.aladdin-e.com). The acrylic acid (AA), the Nile red, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC), N-hydroxysuccinimide (NHS), bovine serum albumin (BSA) and 2-(N-morpholine)-ethane sulphonic acid (MES, ultra-pure grade, 99.0%) were purchased from Sigma-Aldrich (Shanghai, China, //www.sigmaaldrich.com/china-mainland.html

Preparation and physicochemical characterization of nanoparticles

A simple one-step synthesis method was used to produce the core-shell nanoparticles with carboxyl-functionalized surface, the synthetic mechanism illustration was shown in Fig. 1. This one-step synthesis using soap-free emulsion polymerization technique with inexpensive compounds of styrene and acrylic acid, which has significant advantages compared to conventional emulsion polymerization with two or more stages [23]. In this polymerization process, the clear core-shell structure of

Conclusions

A simple, fast, cost-effective and user-friendly method for detecting the CRP has been described. The method was based on the fluorescent carboxyl polystyrene nanoparticle which was synthesized by soap free emulsion polymerization. Under the optimized conditions, the detection limits can reach 0.091 mg/L, the concentration of CRP could be measured in a large dynamic range in plasma (0.1–160 mg/L) within a rapid detection time (3 min), and the precision of the intra-assay and inter-assay was

Acknowledgments

This work is supported by the Program of Guangdong Provincial Science & Technology (No. 2017A020208014). We gratefully acknowledge Dr. Netsanet Shiferaw Terefe from CSIRO for the constructive suggestion and language help.

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