A facile chemiluminescence sensing for ultrasensitive detection of heparin using charge effect of positively-charged AuNPs

https://doi.org/10.1016/j.saa.2019.03.073Get rights and content

Highlights

  • A facile, rapid, ultrasensitive and selective CL sensing for heparin is achieved.

  • The sensing is by heparin's electrostatic action and charge effect of signal probe.

  • The sensitivity is due to heparin's highly negative charge and powerful charge effect.

  • The highest negative charge of heparin led to high selectivity for this CL sensing.

  • Heparin detection in real human serum samples exhibits good reliability.

Abstract

Heparin is a glycosaminoglycan with the highest negative charge density of any known biological molecule. Herein, this highly negative charge structure of heparin and the charge effect from positively-charged AuNPs for luminol chemiluminescence (CL) reaction were combined to build a facile and sensitive CL strategy for detection of heparin. The highly negative charge structure of heparin molecules (four negatively-charged side groups per repeat unit) and the effective signal amplification of charge effect from positively-charged AuNPs make this analysis to display high sensitivity for heparin detection, and the detection limit is as low as 0.06 ng/mL. It is about two orders of magnitude lower than the previously reported colorimetric assay and far lower than the current analysis methods. The established CL strategy is to use the electrostatic interaction between heparin and signal probe (positively-charged AuNPs). Since polyanionic heparin has the highest negative charge in biological system, this CL sensing shows high selectivity for the detection of heparin, and hyaluronic acid (HA), an analogue of heparin, cannot cause interference. This CL sensing succeeded in detecting heparin in human serum samples. Besides, polycationic protamine, heparin antidote, can respond to the system's CL signals through its strong interactions with heparin, thus indirectly detecting protamine. For protamine in serum samples, the detection result was basically consistent with Coomassie brilliant blue assay.

Introduction

Heparin is a naturally occurring and highly sulfated glycosaminoglycan. Since it is capable of accelerating the inactivation rate of coagulation factors such as fibrin-bound thrombin, heparin is widely used as an injectable anticoagulant/antithrombotic agent during clinical procedures such as cardiac/vascular surgery and kidney dialysis [1,2]. But, the overdose or long-term use of heparin can lead to fatal blood effect like hemorrhages and thrombocytopenia [3,4]. So, the real-time continuous measurement of the dose of heparin in serum is of importance for the clinical procedures of surgery and postoperative therapy, and highly sensitively monitoring the levels of heparin within infusion solutions was also very necessary to avoid dangerous human errors in dosing, especially for pediatric patients. Traditional heparin dose detection clinically relies on activated clotting time assay and activated partial thromboplastin time assay [5,6]. These clinical assays are not sufficiently credible and accurate for clinical settings because of their lack of specificity and have potential interference from other factors. Thus, many efforts have attempted to develop new methods for the detection of heparin, including surface enhanced Raman spectroscopy [7], fluorimetry [[8], [9], [10]], colorimetric assay [11,12], and electrochemical methods [13]. These assays promote the research for heparin detection and some even undergo clinical testing. However, their analysis sensitivity is still not satisfactory enough to meet clinical needs, and some methods are tedious, time-consuming and costly to detect. Therefore, the facile analysis technique for heparin detection with high sensitivity which can meet the clinical needs is still urgently needed.

Chemiluminescence (CL) analysis has become the major detection technology due to its remarkable advantages of high sensitivity and simple instrumentation [14,15]. In order to further improve the sensitivity of CL detection, various nano-structure materials including nanoparticles have be involved in the CL sensing analysis system to amplify the detected signals [[16], [17], [18], [19], [20], [21], [22], [23], [24]]. Among them, nanoparticles attracted much attention for their simple preparation and sensitive response. Nanoparticles generally use the labeled or label-free manner to amplify the responded CL signal and improve the sensitivity of the analysis [14,[16], [17], [18], [19],[23], [24], [25]]. In the labeled manner, the nanoparticles are usually labeled on the target or signal probe, and the multi-step label procedures were the dominating way for achieving the ideal amplification effect [[16], [17], [18], [19]]. Obviously, the multi-step label is cumbersome and with heavy labor intensity, which may also produce false signals and affect the accuracy of the assay. Unquestionably, label-free manner is simple and acclaimed. For label-free manner, the idiomatic idea and way is seeking the high efficient catalyst for CL reaction to amplify the target response signal [14,[23], [24], [25]]. In various catalysts used in label-free CL assay strategy, gold nanoparticles (AuNPs) shows its powerful amplifying signal ability [14,23,25]. The CL assay built by means of nanoparticles' physical and chemical effect from surface microstructure were found to be an excellent analysis sensing system, and can be used for simple and high sensitive detection of protein and other targets [14,23,25]. In particular, our previous researches found that the tiny changes in surface charged property of nanoparticles can lead to significant changes in the catalytic performance for CL reaction and this charge effect of nanoparticles displayed high efficiency and attractive sensitivity [14,23]. Based on the charge effect of AuNPs, we successfully constructed the high sensitive and rapid CL detection system for DNA, amino acids and inorganic metal ions [14,23]. At the same time, the CL sensing based on AuNPs' charge effect is proved to be a highly sensitive detection system.

Heparin is an anionic biological molecule with high negative charge (Scheme 1A). Due to exactly the remarkable electric charge structure, various analytical techniques, as shown in Table 1, like fluorescent [26,27], colorimetric [12], light scattering [28], phosphorescent [29] assays and amperometric assay [30] can be constructed through the electrostatic interaction between negatively charged heparin and the positively charged probes. In 2011, Cao et al. reported visual approach to colorimetric detection of heparin based on the electrostatic interaction between the negatively charged heparin and the positively charged AuNPs [12]. Inspired by this research, whether the high negative charge characteristics of heparin structure can be directly combined with the charge effect of AuNPs for CL reaction is the key to the construction of high sensitive CL nanometer sensing to detect heparin. The molecular structure feature of heparin also created the important key conditions for the high sensitive CL detection of heparin based on AuNPs' charge effect. This study just applied the electrostatic interaction between the negatively charged heparin and the positively charged AuNPs to positively-charged AuNPs' charge effect on luminol CL detection in order to design the convenient and rapid CL assay strategy for heparin detection with ultra-high sensitivity. The schematic diagram is shown in Scheme 1B. Positively-charged AuNPs itself possess the high surface positive charge density, and the charge effect can catalyze luminol-hydrogen peroxide CL detection to generate strong CL signal; whereas after the interaction between negatively charged heparin and positively-charged AuNPs, the electropositivity on the surface of positively-charged AuNPs can be shielded by heparin resulting in the generation of weak CL signal. Thus, CL signals respond to heparin. Because that heparin has the highest negative charge density of any known biological molecule [1,2], the proposed CL sensing based on charge effect has high selectivity for heparin detection. The research found that an analogue of heparin, hyaluronic acid (HA), also did not interfere. In this analysis, the sensitive and effective signal amplification from positively-charged AuNPs' charge effect led to the high sensitivity of heparin detection and the detection limit is low to 0.06 ng/mL, which was far lower than that of the previous analysis method [[26], [27], [28]] and was about two orders of magnitude lower than the color analysis based on electrostatic interaction [12]. It's perfectly capable of meeting the clinical needs. Certainly, this developed CL sensing for heparin detection provides an important theoretical basis for clinical diagnosis and monitoring heparin content and unfolds bright prospects for clinical application.

Section snippets

Chemicals and materials

Heparin sodium was obtained from Aladdin Chemistry Co. Ltd. (185 U/mg, Shanghai, China). Heparin stock solution was prepared by dissolving heparin sodium in water and diluting with water to a calibrated flask. The working solution was further diluted with water. Chloroauric acid (HAuCl4), cysteamine and sodium borohydride for synthesis of positively charged AuNPs were purchased from Sigma (St. Louis, MO, USA). Protamine, Chondroitin sulfate (Chs), HA, chitosan, polylysine, glucose, lactic acid

Strategy of this facile CL sensing for detecting heparin

The design strategy of this facile CL sensing for detection of heparin is built based on the electrostatic interaction between negatively-charged heparin and positively-charged AuNPs and positively-charged AuNPs' charge effect on luminol CL reaction. Theoretically, negatively-charged heparin can shield the electropositivity of positively-charged AuNPs' surface through electrostatic attraction, and the changes in electropositivity of the AuNPs surface affect its catalytic performance for luminol

Conclusions

In summary, a facile and sensitive CL analysis strategy for the detection of heparin was developed in this study. This CL sensing has the following outstanding advantages: (1) The method adopts the homogeneous analysis model of “Incubation-CL detection” in liquid phase. So, it is convenient, cheap and practical. (2) The high electronegativity (four negatively-charged side groups per repeat unit) of heparin structure can fully demonstrate the signal amplification of the charge effect from

Acknowledgements

This work was supported financially by the National Natural Science Foundation of China (No.21605018), the Natural Science Basic Research Project of Shaanxi Province of China (No.2018JM5149), the Foundation Research Project of Shaanxi Provincial Key Laboratory of Geological Support for Coal Green Exploitation (No.MTy2019-05), and the Natural Science Foundation of Fujian Province of China (No.2017J01472).

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