A novel fluorescent nanosensor for detection of heparin and heparinase based on CuInS₂ quantum dots

Highlights

• A novel fluorescence nanosensor for detection of heparin and heparinase was established.

• The method was based on the fluorescence “turn-off” and “turn-on” of CuInS₂ QDs.

• Good sensitivity and selectivity were obtained for the determination of heparin.

• Heparin in real sample was detected with satisfactory results.

Abstract

In this work, a novel fluorescence “turn off–on” nanosensor for the determination of heparin and heparinase based on CuInS₂ quantum dots (QDs) was established. CuInS₂ QDs (modified by l-cysteine) featuring amino groups were directly prepared in aqueous solution via a hydrothermal synthesis method. The amino groups on the surface of CuInS₂ QDs can interact with sulfate and carboxylate groups in heparin via electrostatic interactions and hydrogen bonding, which led the fluorescence of CuInS₂ QDs to “turn-off”. However, the heparin could be hydrolyzed into small fragments in the presence of heparinase, which resulted in the fluorescence of CuInS₂ QDs being recovered. Therefore, the addition of heparinase to the heparin/CuInS₂ QDs system activated the fluorescence of CuInS₂ QDs to “turn-on” state. Thus, the determination of heparin and heparinase could be achieved by monitoring the fluorescence “turn off–on”. Under the optimum conditions, there was a good linear relationship between I/I₀ (I and I₀ were the fluorescence intensity of CuInS₂ QDs in the presence and absence of heparin, respectively) and heparin concentration in the range of 0.05–15 μmol L⁻¹ with the detection limit of 12.46 nmol L⁻¹. The linear detection for heparinase was in the range of 0.2–5 μg mL⁻¹ with the detection limit of 0.07 μg mL⁻¹. The proposed nanosensor was employed for the detection of heparin in fetal bovine serum samples with satisfactory results.

Introduction

Heparin, consisting of repeating units of 1→4-linked pyranosyluronic acid and 2-amino-2-deoxyglucopyranose residues, is a highly sulfated linear acidic polysaccharide belonging to the category of glycosaminoglycans (GAG) (Caplia and Linhardt, 2002, Whitelock and Iozzo, 2005). Known as the most negatively charged biological macromolecule (Rabenstein, 2002, Linhardt and Toida, 2004), heparin plays a significant role in the regulation of various physiological processes, especially as a major clinical anticoagulant drug to prevent thrombosis during surgery and to treat thrombotic diseases. However, heparin overdose could induce thrombocytopenia, which is recognized as one of the most catastrophic complications of heparin treatment (Hirsh et al., 1992; Girolami and Girolami, 2006). The proper therapeutic dosing level of heparin is 2–8 U mL⁻¹ (17–67 μmol L⁻¹) during cardiovascular surgery and 0.2–1.2 U mL⁻¹ (1.7–10 μmol L⁻¹) for postoperative and long-term therapy (Zhan et al., 2010, Hirsh and Raschke, 2004). Therefore, close monitoring and quantification of heparin in serum during the surgery and the anticoagulant therapy period is of crucial significance.

Up to now, there are many assays for heparin quantification and/or detection. Traditional laboratory assays for heparin quantification are indirect, which rely on monitoring of the activated coagulation time (ACT) or the activated partial thromboplastin time (aPTT) (Murray et al., 1997). These assays are not sufficiently accurate and reliable because of their lack of specificity and potential interference from other factors (Levine et al., 1994). Recently, a wide variety of fluorescent and colorimetric methods have been established for heparin sensing (Wright et al., 2005, Mecca et al., 2006, Briza et al., 2008, Sun et al., 2007, Sauceda et al., 2007; Egawa et al., 2008, Zhong and Anslyn, 2002). Synthetic cationic chromophores, such as tripodal boronic acids (Wright et al., 2005), polycationic calyx [8] arenes (Mecca et al., 2006), polymethinium salts (Briza et al., 2008), and a chromophore-tethered flexible copolymer (Sun et al., 2007), have been used as heparin indicators. Most of these assays adopt fluorescence quenching as signal output. Subsequently, a peptide based sensor was reported to show fluorescence increase upon interaction with heparin (Sauceda et al., 2007). However, the detection window of the fluorescence turn on assay (0–0.4 U/mL) is out of the clinical range. More recently, two benzothiadiazole (BT) containing conjugated polymers were used for naked-eye detection and quantification of heparin (Pu and Liu, 2008, Tang et al., 2006). Although both polymers are able to quantify purified heparin in nearly the whole clinical detection range, they are not suitable for heparin sensing in plasma or serum as the laborious steps and the polymer fluorescence is greatly affected by proteins in these biological media (Zhan et al., 2010). It is highly desirable to develop new assays for convenient and selective detection and quantification of heparin in complex biological media.

Heparinase, a heparin-degrading enzyme produced by Flavobacterium heparinum, is used to deheparinize blood following extracorporeal procedures in surgery and in other applications (Sasisekharan et al., 1995). Heparinases can specifically cleave the glucosamine (1→4) uronsy linkage present in the heparin polymer. Enzymatic degradation of heparin by heparinases has not only largely facilitated heparin structural analysis and contamination detection, but also showed great potential to be a green and cost-effective way to produce low molecular weight heparin.¹ Besides, heparinase affects various biological processes including morphogenesis, angiogenesis, inflammation, tumor invasion, and metastasis (Chen et al., 2007). Obviously, the detection of heparinase is also quite meaningful while the assay for the determination of heparinase still remains rare.

Quantum dots (QDs) are semiconductor nanocrystals with unique electro-optical properties (Ma and Su, 2011), such as high quantum yields, long fluorescence lifetimes, large extinction coefficients, pronounced photostability, and broad absorption spectra coupled with narrow photoluminescent (PL) emission spectra (Mattoussi et al., 1999; Jaiswal and Simon, 2004). There were some reports on the fluorescence turn-on and turn-off of QDs applied to the determination (Liu et al., 2012a, Liu et al., 2012b, Chen et al., 2013, Zhang et al., 2008, Noh et al., 2010, Gao et al., 2012a, Gao et al., 2012b). However, the applications of QDs to the clinical field have been hampered owing to the high toxicity of the QDs. So far the majority of the reports focused on the cadmium-based QDs that are toxic to biological systems, sensitive to heat, chemical and photochemical disturbances, and eventually would cause serious environmental problems due to the leakage of cadmium (Bagalkot et al., 2007, Cho et al., 2007, Pradhan et al., 2005, Liu et al., 2013). With regard to the toxicity of QDs consisting of cadmium element, there are two main approaches to reduce the toxicity of QDs. One is to cover non-toxic substance, such as silica shell (Wang et al., 2009a, Wang et al., 2009b, Cao et al., 2006, Cho et al., 2010) and the other is to develop novel QDs without heavy metal ions(Wang et al., 2009a, Wang et al., 2009b, Yi et al., 2001, Lan et al., 2007, Niu et al., 2012). In recent years, the synthesis and application of I–III–VI CuInS₂ QDs that does not contain any toxic class A elements (Cd, Pb, and Hg) or class B elements (Se and As) have attracted considerable attention (Castro et al., 2004, Nakamura et al., 2006, Pan et al., 2008, Xie et al., 2009, Liu et al., 2013). In this work, we used the l-cysteine capped CuInS₂ QDs which are a novel class of toxic heavy metal-free emitters.

CuInS₂ QDs, as a near-infrared (NIR) region QDs (generally wavelengths>650 nm) (Gao et al., 2012a, Gao et al., 2012b), have attracted more attention. Because biosensors operating in the NIR region can avoid interference from biological media, mainly tissue autofluorescence and scattering light, they can facilitate relatively interference-free sensing. Although great explorations of NIR fluorescence sensors have been made in the field of biological imaging, the NIR fluorescence sensors for the determination of biomolecules are quite few (Zhang et al., 2009; Jeng Esther et al., 2006, Liu et al., 2013).

Herein we report a novel and simple fluorescence nanosensor for both heparin and heparinase detection by taking advantage of the fluorescence “turn-off” and “turn-on” feature of CuInS₂ QDs. The water-soluble CuInS₂ QDs (modified by l-cysteine) featuring amino groups were directly prepared in aqueous solution via a hydrothermal synthesis method (Liu et al., 2012a, Liu et al., 2012b). The amimo groups on the surface of CuInS₂ QDs can interact with sulfate and carboxylate groups in heparin via electrostatic interactions and hydrogen bonding to form a CuInS₂ QDs/heparin complex, which will lead to the quenching of the fluorescence intensity of CuInS₂ QDs. Once heparinase is introduced into the CuInS₂ QDs/heparin system, it can hydrolyze the heparin into small fragments with low charge density, and the fluorescence of CuInS₂ QDs would be recovered. To the best of our knowledge, the fluorescence “turn off–on” method for heparin and heparinase detection based on NIR QDs have not been reported before.