Elsevier

Biosensors and Bioelectronics

Volume 86, 15 December 2016, Pages 109-114
Biosensors and Bioelectronics

Rapid and highly-sensitive uric acid sensing based on enzymatic catalysis-induced upconversion inner filter effect

https://doi.org/10.1016/j.bios.2016.06.017Get rights and content

Highlights

  • A novel fluorescent nanosensor for detection of uric acid (UA) is developed.

  • This sensor is based on enzymatic catalysis-induced upconversion inner filter effect.

  • The new biosensor was successfully applied to human serum samples.

Abstract

In this work, a novel and simple fluorescence method for detection of uric acid (UA) based on NaYF4:Yb3+, Tm3+ upconversion nanoparticles (UCNPs) is developed. The proposed method is based on the fact that uricase can oxidize uric acid to allantoin and hydrogen peroxide, which, on its turn, can oxidize o-phenylenediamine (OPD) to the oxidized OPD (oxOPD). The fluorescence of UCNPs can be significantly quenched by oxOPD through inner filter effects (IFE). Under the optimized conditions, the UA concentration was proportional to the changes in fluorescence intensity of UCNPs. A linear response was obtained over the concentration range from 20 to 850 μΜ with the low detection limit of 6.7 μΜ for uric acid. More importantly, this method has the potential to detect uric acid in human serum samples, suggesting the nanosensor can be used in a complex biological sample matrix.

Introduction

Rare-earth (RE) doped upconversion nanoparticles (UCNPs) have attracted considerable attention. Upconversion nanoparticles can transform the long wavelength radiation to the short wavelength radiation via a two-photon or multiphoton mechanism (Deng et al., 2011, Han et al., 2014, Zhou et al., 2014a, Zhou et al., 2014b). Comparing with organic fluorophores or other nano flurourence probe, UCNPs possess several outstanding features: (1) they can be excited by low power continuous wavelength lasers, which produce less damage to biological samples; (2) they offer narrow emission peaks, large Stokes shifts, low toxicity, high photostability, and thermal stability; (3) the NIR excitation source (typically 980 nm) offers greater tissue penetration depth than conventional ultraviolet excitation; (4) the NIR-excitation technique features nonblinking and non-autofluorescence, what's more, it provides an enlarged signal-to-background ratio and improved sensitivities (Chen et al., 2014). To date, these excellent fluorescence properties make the UCNPs widely applied in biolabeling, biological imaging, and biosensing (Mader et al., 2010; Deng et al., 2011, Peng et al., 2011, Wang et al., 2011, Tang et al., 2013, Chen et al., 2014, Ma et al., 2014, Yao et al., 2014, Zhou et al., 2014a, Zhou et al., 2014b), which have been regarded as decent substitutes for traditional organic fluorescent dyes in fluorimetry.

There are many researches about fluorimetric methods for detection of various analytes based on fluorescence resonance energy transfer (FRET) between UCNPs (donor) and the accepter that have been reported (Rocha et al., 2010; Moghadam et al., 2011; Wu et al., 2013, Wu et al., 2015). However, the formation of FRET process requires a particular distance (generally less than 10 nm) between UCNPs (donors) and accepters (Lin et al., 2013). Thus, in order to ensure the valid interaction, the establishment of the FRET-based sensors above commonly demands complex surface chemical modification of donors and accepters, which would distinctly undermine their practicality. The inner filter effect (IFE) should be able to solve this problem. The inner filter effect (IFE) is an important model and one of the non-irradiation energy conversion models in spectrofluorometry, which results from the absorptions of the excitation and emission light or both simultaneously by absorbents in the detection system (Xu et al., 2011, Cao et al., 2014, Yan et al., 2014). Compared to other fluorescence analysing systems, the inner filter effect (IFE) system possesses several advantages, such as the simplified synthesis and modification process of the fluorescent materials, no covalent linking between the energy receptor and donor, and higher sensitivity of the fluorescent analysis (Li et al., 2007; Wang et al., 2007; Zhang et al., 2012). There were almost no report on the sensitive detection of various analytes associated with the inner filter effect by using label-free UCNPs. Therefore, the development of a novel fluorescent biosensor based on inner filter effect (IFE) using UCNPs without background interference is of great significance.

Uric acid (2,6,8-trihydroxypurine, UA) is the principal end product of purine metabolism in human urine (Huang et al., 2014) and its determination serves as an important marker molecule for the detection of diseases associated with purine metabolism, leukemia pneumonia and variations urate level in body fluids (e.g. serum and urine) (Azmi et al., 2015, Kumar et al., 2014). Generally, the normal level of uric acid is within the range of 0.13-0.46 mM (2.18-7.7 mg/dL) in serum and 1.49-4.46 mM (25-74 mg/dL) in urinary excretion (Azmi et al., 2015). Extreme high level of UA in the blood, known as hyperuricemia or Lesch-Nyhan syndrome, is linked with diseases, such as gout, high cholesterol, high blood pressure, kidney disease, renal impairment and cardiovascular diseases (Piermarini et al., 2013, Wang et al., 2013, Kumar et al., 2014, Zhang et al., 2015). Reversely, abnormally low uric acid levels will also cause oxidative stress and multiple sclerosis in humans (Ghosh et al., 2015). Hence, the quantitative determination of UA at various levels is of great significance in diagnosing of related diseases.

Nowadays, various analytical techniques have been developed for the detection of UA. Despite tremendous efforts was made to detection of UA with high selectivity and sensitivity (Dai et al., 2007, Zuo et al., 2011, Huang et al., 2014, Wang et al., 2014, Arora et al., 2009; Wang et al., 2012; Bera et al., 2011, Ballesta-Claver et al., 2013, Yao et al., 2003, Öhman, 1979), these methods still involved sophisticated equipment, complicated sample preparation processes, high cost, and time consuming immobilizing processes, which make them unsuitable for rapid detection and restrain their wide applications. The florescence method in detection of UA is fast, efficient and sensitive. Recently, a simple and sensitive fluorescence method based on biosensor for the determination of uric acid using H2O2-sensitive quantum dots/dual enzymes has been developed (Azmi et al., 2015). However, it is difficult to eliminate the background interference resulting from the detection systems (e.g. serum and urine), which affects the sensitivity and detection limit. Therefore, it is necessary to develop simple, fast, accurate and sensitive fluorescence methods for the determination of uric acid without background interference.

In this study, we have developed a new UCNPs-based optical biosensor for uric acid determination for the first time. The biosensing system results from enzymatic reaction of uricase and HRP (horseradish peroxidase) which involves oxidizing uric acid to allaintoin and hydrogen peroxide. Hydrogen peroxide can oxidize o-phenylenediamine (OPD) to induce a yellow color and an absorption peak centered at 450 nm. The fluorescence of UCNPs can be significantly quenched by oxOPD through inner filter effects (IFE), which was proportional to uric acid. To our knowledge, there are no previous reports on the combination of UCNPs with uricase/HRP enzymes for uric acid determination. Based on these facts, we demonstrate a simple, fast, sensitive and reliable UA sensing system. The proposed detection strategy was shown in Scheme 1.

Section snippets

Materials

Rare earth oxides, including Y2O3, Yb2O3, Tm2O3, were purchased from China National Pharmaceutical Group Corporation (Shanghai, China). Uric acid, peroxidase, and uricase were obtained from sigma-aldrich. Ascorbic acid, urea, NaCl, cysteine, KCl, glutathione, cholesterol, and glucose were purchased from beijing chemical corp. All the other chemicals (99%, Merck) used in this work were of analytical grade and millipore milli-Q ultrapure water was used throughout the experiments. The ohmic

Characterization of the UCNPs

The morphology, structure, and optical properties of UCNPs were characterized, and the results are shown in Fig. 1. The TEM results (Fig. 1A) revealed that the obtained UCNPs are uniform in size and morphology, and are well-dispersed with the average size of 40 nm. As shown in Fig. 1B, NaYF4:Yb, Tm nanocrystals were obtained consisting of a dominant cubic phase (JCPDS no.77-2042) by the XRD pattern, indicating the highly crystalline nature of the synthesized material. FT-IR spectrum was used to

Conclusion

In conclusion, we have developed a novel, convenient, sensitive and selective fluorescence assay for the quantitative detection of uric acid based on the quenching of the fluorescence of UCNPs. This fluorescence method offered several distinct advantages: (1) the superiority of this approach lies in that it does not require specific modification; (2) the reaction was very fast, and thus the detecting process becomes more flexible, fast and simple; (3) in a simple manner it can be used for uric

Acknowledgments

This work was supported by the National Natural Science Foundation of China (21375037, 21275051 and 21475043), Scientific Research Fund of Hunan Provincial Science and Technology Department and Education Department (13JJ2020, 12A084), and Doctoral Fund of Ministry of Education of China (NO: 20134306110006).

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