Analytica Chimica Acta 883 (2015) 32–36

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Laser-induced fluorescence reader with a turbidimetric system for sandwich-type immunoassay using nanoparticles Kim Y.H., Lim H.B. * Department of Chemistry, Dankook University, 126 Juckjeon-dong, Suji-gu, Yongin-si, Gyeonggi-do 448-701, Republic of Korea

H I G H L I G H T S

G R A P H I C A L A B S T R A C T


Laser-induced fluorescence system with ratiometric correction was developed.
The system reduced experimental error caused by particle loss and aggregation.
The detection limit of about 39 pg mL1 for salinomycin was obtained.
Calibration linearity and sensitivity were also significantly improved.
The system has the potential for bioanalysis using various nanoparticles.

Laser-induced fluorescence reader with ratiometric correction for sandwich-type immunoassay using nanoparticles.

A R T I C L E I N F O

A B S T R A C T

Article history: Received 9 March 2015 Accepted 14 April 2015 Available online 20 April 2015

A unique laser-induced fluorescence (LIF) reader equipped with a turbidimetric system was developed for a sandwich-type immunoassay using nanoparticles. The system was specifically designed to reduce experimental error caused by particle loss, aggregation and sinking, and to improve analytical performance through ratiometric measurement of the fluorescence with respect to the turbidimetric absorbance. For application to determine the concentration of salinomycin, magnetic nanoparticles (MNPs) and FITC-doped silica nanoparticles (colored balls) immobilized with antibody were synthesized for magnetic extraction and for tagging as a fluorescence probe, respectively. The detection limit of about 39 pg mL1 was obtained, which was an improvement of about 2-fold compared to that obtained without employment of the turbidimetric system. Calibration linearity and sensitivity were also improved, with increase from 0.8601 to 0.9905 in the R2-coefficient and by 1.92-fold for the curve slope, respectively. The developed LIF reader has the potential to be used for fluorescence measurements using various nanomaterials, such as quantum dots. ã 2015 Elsevier B.V. All rights reserved.

Keywords: Sandwich-type immunoassay Laser-induced fluorescence Nanoparticles Ratiometric correction Absorbance

1. Introduction

* Corresponding author. Tel.: +82 31 8005 3151. E-mail address: [email protected] (H.B. Lim). http://dx.doi.org/10.1016/j.aca.2015.04.031 0003-2670/ ã 2015 Elsevier B.V. All rights reserved.

Recently, immunoassay using magnetic nanoparticles has been extensively applied for the determination of various bio-targets, such as antibiotics, biomarkers, cells, etc. High-pressure liquid

Y.H. Kim, H.B. Lim / Analytica Chimica Acta 883 (2015) 32–36

chromatography (HPLC) [1–3], liquid chromatography–mass spectrometry (LC–MS) [4,5], enzyme-linked immunosorbent assay (ELISA) [6–8], chemiluminescence (CL) [9–13], and fluorescence [14–18] are the common detection techniques employed in such immunoassays. Among them, HPLC and LC–MS are accompanied with the advantages of high sensitivity, low limit of detection (LOD) and high resolution. However, these methods require high cost for maintenance, in addition to sophisticated operation and expertized sample pre-treatment [16]. While chemiluminescence provides the merit of high sensitivity owing to extremely low background, it also suffers from poor stability [19–21]. Compared to these methods, ELISA provides the advantages of portability and speed, but suffers from relatively poor detection limit. Recently, a new analytical platform for the determination of various antibiotics from food products was introduced, which employed nanoparticles and laser-induced florescence (LIF) spectrometry [16]. According to the measurement platform, MNPs were used to extract antibiotic targets by magnetic separation, after which tagging was carried out with colored balls as a probe for detection. The platform showed excellent sensitivity and selectivity owing to the signal amplification and immunoassay. In addition, the colored balls allowed the fluorescent measurement to be relatively free from quenching and photo bleaching [22,23]. However, this method suffered from poor particle stability and dispersion due to aggregation and sinking during the treatment and measurement process. Although many fluorescence techniques using various types of nanoparticles have been developed so far, no report has tackled these issues instrumentally. In this work, an LIF reader equipped with a turbidimetric system was built in order to measure the concentration of an antibiotic by a sandwich-type immunoassay using nanoparticles. Since the immunoassay platform required centrifugation and magnetic separation, followed by washing, the analytical results suffered from particle loss which occurred during these steps. Therefore, the sample extraction process requires careful treatment throughout the experiment. Furthermore, as mentioned above, the extracted products dispersed in a sample cell can become aggregated or sink during the LIF measurement, causing signal reduction or fluctuation. Such issues can be minimized by a ratiometric measurement using the turbidimetric system attached to the LIF reader in the developed system, i.e., the fluorescence intensity divided by the turbidimetric absorbance would allow reflection of the number of particles effectively being measured. Although ratiometry is employed in various analysis methods, turbidimetric correction has not yet been introduced for the sandwich-type immunoassay using nanoparticles. The performance of the developed system was demonstrated by determining the concentration of salinomycin, which is a polyether

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ionophore antibiotic effective for feed and growth facilitation [24]. Since its misuse induced serious intoxication, causing various clinical signs in humans and animals, such as nausea, diarrhea, vomiting, etc. [24], the EU officially prohibited its use. Therefore, the developed LIF system will be useful for market surveillance of food products, and for the study of pharmacokinetics and human toxicity [24]. 2. Experimental The fluorescence reader was designed to measure the fluorescence emitted from the colored nanoparticles tagged on the antibiotic target. For demonstration, salinomycin concentration was determined by a sandwich-type immunoassay platform using two synthesized nanoparticles, i.e., magnetic nanoparticles for target extraction, and dye-doped silica nanoparticles as probes. Improvement of the analytical performance was illustrated by obtaining the analytical figures-of-merit. 2.1. Instrumental design of fluorescence reader The schematic diagram of the designed fluorescence reader is illustrated in Fig. 1. A DPSS diode laser (MBL-III-473-5 mW, CNI, China) was used as the excitation source for fluorescence, with a broadband dielectric mirror (BB1-E02, 400–750 nm, Thorlabs, USA). A cylindrical-shaped quartz detection cell equipped with a reflection cap was used so that the laser beam could multiply pass through the cell from bottom to top, resulting in increase of the cell pathlength. The photons emitted from the probe nanoparticles were detected by the photosensor (H10769PA-40, Hamamatsu, Japan), which was positioned at a right angle, and counted by a photon counting unit (C9744 and C8855-01, Hamamatsu, Japan) after passing through a plano-convex lens (LA1131, f = 50 mm, uncoated, Thorlabs, USA) and an interference filter (FF03-525/30-25, Semrock, USA) for wavelength selection (F). For the measurement of turbidimetric absorbance, a D2 lamp (DO 650 TJ, Heraeus Noblelight) was used for the light source, with a monochromator (Lambda 25) for wavelength selection, and an Si photodiode detector (S1336-44BQ, Hamamatsu, Japan) (A). For data handling and operation, software was designed by Sensor Tech (Sungnam, Korea), through which the measured counts per second were divided by the absorbance for ratiometric treatment. 2.2. Preparation of amine-functionalized Fe3O4 magnetic nanoparticles (MNPs) for target extraction Amine-functionalized Fe3O4 MNPs were synthesized through the alkaline co-precipitation of FeCl36H2O and FeCl24H2O [25],

Fig. 1. Schematic diagram of the laser-induced fluorescence (LIF) reader system (F) combined with turbidimetric absorption measurement (A).

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the procedure for which was previously optimized in our lab. In brief, FeCl24H2O (0.5 g) and FeCl36H2O (1.35 g) were dissolved and mixed in a round bottom flask containing 25 mL of deionized water in an Ar gas flow. Next, 12.5 mL of ammonium hydroxide (28–30%, Sigma–Aldrich Chemical Co.) at 80  C was added, and the mixture was heated for 20 min. A silica layer was then coated on the particles by the addition of 200 mL tetraethyl orthosilicate (TEOS, 99.999%, Sigma–Aldrich Chemical Co.) into 100 mL of ethanol containing 100 mg of the MNPs after adjustment of the pH to 9. The silica layer was functionalized with amine groups through reaction with 1 mL of 3-(aminopropyl)triethoxysilane (APTEOS, 99%, Sigma–Aldrich Chemical Co.) for 24 h, and then washed three times with acetone and stored in ethanol for the next step. 2.3. Preparation of FITC-doped silica NPs (colored balls) as a probe and subsequent surface functionalization and immobilization FITC-doped colored balls were prepared by the reverse microemulsion method, which was also previously optimized in our lab. In detail, FITC–silica cores were synthesized by the reaction of 10 mg FITC (90%, Sigma–Aldrich Co.) in 10 mL ethanol with 50 mL APTEOS for 24 h in the dark. To prepare the precursor, approximately 5 mmol docusate sodium salt (Sigma ultra, 99%, Sigma–Aldrich Co.) and 860 mL deionized water were added to 48 mL heptane (Anhydrous, 99%, Sigma–Aldrich Co.) and stirred for 10 min. After 300 mL of precursor was added and aged for 30 min, 430 mL of TEOS (99.99%, Sigma–Aldrich Co.) and 260 mL of ammonium hydroxide (ACS reagent, 28–30%, Sigma–Aldrich Co.) were added and aged again in the dark for 12 h. The colored balls were then separated and purified three times by centrifugation (Gyro 416G, Gyrozen Co.) for 10 min at 4000 rpm and washing with ethanol. The presence of FITC at the core was confirmed by the green fluorescence image obtained for LIFM. Functionalization of the nanoparticles with an amine group was done by reacting with 50 mL of APTEOS for 24 h, followed by purification and washing three times. For immobilization of the salinomycin antibody, 10 mL IgG (Polyclonal, sheep, Abcam, UK) was added and reacted with a mixture (2:1) of N-(3-dimethylaminopropyl)-N-ethylcarbodiimide (0.2 mmol) (EDC, 99%, Sigma–Aldrich Co.) and N-hydroxysuccinimide (0.1 mmol) (NHS, 99%, Sigma–Aldrich Co.) for 24 h, followed by washing three times.

2.4. Extraction of antibiotics using amine-functionalized magnetic NPs Standard salinomycin solutions of 0.2, 0.5, 1, 2, 5, and 10 ng mL1 were prepared by dissolving salinomycin monosodium salt hydrate (Sigma–Aldrich Co.) in methanol (HPLC, 99.9%, Sigma–Aldrich Co.). Next, 1.725 mg of MNPs were added and reacted with a 2:1 mixture of EDC (0.2 mmol) and NHS (0.1 mmol) for 2 h. The final products were washed three times with phosphate-buffered saline (PBS) (1, Sigma–Aldrich Co.) and collected through magnetic separation. The two types of nanoparticles prepared were reacted for 30 min at room temperature, producing a sandwiched product. The concentration of salinomycin was then determined by the LIF reader. For ratiometric measurement, fluorescence intensity and absorbance were measured at the same experimental conditions. 3. Results and discussion 3.1. Analytical platform for the determination of salinomycin Herein, an LIF reader equipped with a turbidimetric system was applied to determine the concentration of salinomycin by use of the sandwich-type immunoassay platform, illustrated in Fig. 2. As shown in the figure, the antibiotic target was extracted by MNPs and tagged with FITC-doped color balls through immunoreaction for fluorescence detection. Unlike a typical sandwich-type platform, the target was directly bound to the MNPs through the formation of an amide bond, which enhanced the stability of the product and minimized target loss during the washing steps. Selectivity of the platform was achieved through the immunoreaction of the tagging. 3.2. Synthesis of MNPs and amine functionalization For efficient target extraction, the MNPs should be sufficiently dispersed and small in size to react with the antibiotics. SEM images of the synthesized Fe3O4 MNPs and colored balls are shown in Fig. 3. The average size of the amine-functionalized MMPs was 25.1  4.7 nm (Fig. 3(a)), which was adequate for maintenance of the superparamagnetic property (> ec, ea, and es, as shown in Supplementary Fig. S5, indicating that the measured absorbance, A, was mostly influenced by the number of MNPs, nM, at 365 nm. Therefore, the ratiometric measurement of If/A can be used to minimize the measurement error caused by MNP motions, i.e.,

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Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2015.04.031. References

Fig. 5. Calibration curves of salinomycin using the sandwiched nanoparticle immunoassay platform: (a) without ratiometric correction, and (b) with ratiometric correction (normalized).

particle loss, sinking, and aggregation. When this ratiometric method was applied to the determination of salinomycin (n = 6) spiked in phosphate buffered saline solution (X1, Sigma–Aldrich Co.), the linearity of the calibration curve (R2: linear regression coefficient) was significantly improved from 0.8601 to 0.9905, as expected. In addition, the calibration sensitivity (slope) was also enhanced by 1.9 times when normalized at 0.1 ng mL1, as shown in Fig. 5. From the calibration curve (3s /m), the detection limit was determined to be 39 pg mL1, which was about 2 times better than that obtained without use of the turbidimetric system. The binding ratio of MNP can be one of the clues to explain this result. Since the optimized ratio of MNP with respect to salinomycin was 0.7, the fluorescence intensity was more sensitive to particle loss than the absorbance of MNP. In addition, the probe nanoparticles reacted with salinomycin will have more chance to be lost during the treatment and measurement process. Therefore, once particle loss or sinking was occurred, it seemed that the reduction of fluorescence intensity became larger than that of turbidimetric absorbance. The obtained detection limit was about 1.5  102 times higher than that of LC–MS/MS, but about 1.2  103 times lower than that of ELISA. Conclusively, the ratiometric correction using the turbidimetric system improved the linearity of the calibration curve, while also increasing the calibration sensitivity in this work. 4. Conclusions The developed fluorescence reader equipped with a turbidimetric system for ratiometric measurement performed excellently for the determination of salinomycin concentration using the sandwiched immunoassay platform with MNPs. When the experimental parameters were optimized, the detection limit and calibration sensitivity were improved by 2 and 1.9-fold, respectively, compared to those obtained without ratiometric correction. The most remarkable improvement was in the linearity of calibration curve (R2), which was increased from 0.8601 to 0.9905. This technique has the potential to be applied to analytical methods which employ sandwich-type immunoassay with nanoparticles. Acknowledgements This work was supported by the Nano-Convergence Foundation (Development of immuno-biomaterial analyzer using engineered nanomaterials), funded by the Ministry of Science, ICT and Future Planning (MSIP, Korea), and by the Ministry of Trade, Industry and Energy (MOTIE, Korea).

[1] M. Gratacós-Cubarsí, J.A. García-Regueiro, M. Castellari, Assessment of enrofloxacin and ciprofloxacin accumulation in pig and calf hair by HPLC and fluorimetric detection, Anal. Bioanal. Chem. 387 (2006) 1991–1998. [2] N. Phonkeng, R. Burakham, Signal derivatization for HPLC analysis of fluoroquinolone antibiotic residues in milk products, Chromatographia 75 (2012) 233–239. [3] B.C. McWhinney, S.C. Wallis, T. Hillister, J.A. Roberts, J. Lipman, J.P.J. Ungerer, Analysis of 12 beta-lactam antibiotics in human plasma by HPLC with ultraviolet detection, J. Chromatogr. B 878 (2010) 2039–2043. [4] G. Stubbings, T. Bigwood, The development and validation of a multiclass (LC– MS/MS) procedure for the determination of veterinary drug residues in animal tissue using a QuEChERS approach, Anal. Chim. Acta 637 (2009) 68–78. [5] D.G. Kennedy, R.J. McCracken, A. Cannavan, S.A. Hewitt, Use of liquid chromatography mass spectrometry in the analysis of residues of antibiotics in meat and milk, J. Chromatogr. A 812 (1998) 77–98. [6] H. Watanabe, A. Satake, Y. Kido, A. Tsuji, Monoclonal-based enzyme-linked immunosorbent assay and immunochromatographic rapid assay for salinomycin, Anal. Chim. Acta 437 (2001) 31–38. [7] A.C. Huet, C. Charlier, S.A. Tittlemier, G. Singh, S. Benrejeb, P. Delahaut, Simultaneous determination of (fluoro) quinolone antibiotics in kidney, marine products, eggs, and muscle by enzyme linked immunosorbent assay (ELISA), J. Agric. Food Chem. 54 (2006) 2822–2827. [8] V. Andreu, C. Blasco, Y. Pico, Analytical strategies to determine quinolone residues in food and the environment, Trends Anal. Chem. 26 (2007) 534–556. [9] X. Tao, M. Chen, H. Jiang, J. Shen, Z. Wang, K. Wen, X. Wang, X. Wu, Chemiluminescence competitive indirect enzyme immunoassay for 20 fluoroquinolone residues in fish and shrimp based on a single-chain variable fragment, Anal. Bioanal. Chem. 405 (2013) 7477–7484. [10] S. Bi, Y. Yan, X. Yang, S. Zhang, Gold nanolabels for new enhanced chemiluminescence immunoassay of alpha-fetoprotein based on magnetic beads, Chem. Eur. J. 15 (2009) 4704–4709. [11] T. Tanaka, T. Matsunaga, Fully automated chemiluminescence immunoassay of insulin using antibody-protein A-bacterial magnetic particle complexes, Anal. Chem. 72 (2000) 3518–3522. [12] X. Yang, Y. Guo, A. Wang, Luminol–antibody labeled gold nanoparticles for chemiluminescence immunoassay of carcinoembryonic antigen, Anal. Chim. Acta 666 (2010) 91–96. [13] K. Wutz, R. Niessner, M. Seidel, Simultaneous determination of four different antibiotic residues in honey by chemiluminescence multianalyte chip immunoassays, Microchim. Acta 173 (2011) 1–9. [14] P. Peippo, V. Hagren, T. Lovgren, M. Tuomola, Rapid time-resolved fluoroimmunoassay for the screening of narasin and salinomycin residues in poultry and eggs, J. Agric. Food Chem. 52 (2004) 1824–1828. [15] S.J. Kim, J.A. Ko, H.B. Lim, Application of magnetic and core–shell nanoparticles to determine enrofloxacin and its metabolite using laser induced fluorescence microscope, Anal. Chim. Acta 771 (2013) 37–41. [16] J.Y. Park, H.B. Lim, Sample treatment platform using nanoparticles to determine salinomycin in flesh and meat, Food Chem. 160 (2014) 112–117. [17] P. Peippo, V. Hagren, T. Lovgren, M. Tuomola, Rapid time-resolved fluoroimmunoassay for the screening of narasin and salinomycin residues in poultry and eggs, J. Agric. Food Chem. 52 (2004) 1824–1828. [18] J. Chen, F. Xu, H. Jiang, Y. Hou, Q. Rao, P. Guo, S. Ding, A novel quantum dot based fluoroimmunoassay method for detection of enrofloxacin residue in chicken muscle tissue, Food Chem. 113 (2009) 1197–1201. [19] M. Yang, Y. Kostov, H.A. Bruck, A. Rasooly, Gold nanoparticle based enhanced chemiluminescence immunosensor for detection of staphylococcal enterotoxin B (SEB) in food, Int. J. Food Microbiol. 133 (2009) 265–271. [20] C. Yang, Z. Zhang, Z. Shi, A novel chemiluminescence reaction system for the determination of lincomycin with diperiodatonickelate(IV), Microchim. Acta 168 (2010) 293–298. [21] T. Li, E. Wang, S. Dong, Chemiluminescence thrombin aptasensor using high activity DNAzyme as catalytic label, Chem. Commun. 43 (2008) 5520–5522. [22] J.A. Ko, H.B. Lim, Core–shell silica nanoparticles synthesized for quantitative study of DNA cleavage by laser-induced fluorescence microscopy, Anal. Bioanal. Chem. 399 (2011) 1683–1688. [23] J.A. Ko, H.B. Lim, Fluorescence quenching of dye-doped core–shell nanoparticles by metal ions, Anal. Methods 4 (2012) 2009–2012. [24] Y. Li, J. Fang, S. Wu, K. Ma, H. Li, X. Yan, F. Dong, Identification and quantification of salinomycin in intoxicated human plasma by liquid chromatographyelectrospray tandem mass spectrometry, Anal. Bioanal. Chem. 398 (2010) 955–961. [25] J.H. Jang, H.B. Lim, Characterization and analytical application of surface modified magnetic nanoparticles, Microchem. J. 94 (2010) 148–158.