A wide dynamic range detection of biopolymer medicines with resonance light scattering and absorption ratiometry

Abstract

Using the interaction of heparin with Janus Green Blue (JGB) as an example, herein a dual-wavelength resonance lighting scattering (RLS) ratiometric method and an absorbance ratiometric one are developed to detect biopolymer medicines based on their bindings with organic dyes. In aqueous solution, heparin could interact with JGB, displaying significantly enhanced RLS signals in UV–vis region. By measuring the RLS intensity ratio (R_ratio) and absorbance ratio (A_ratio) at the wavelengths of 285 nm over that at 345 nm, respectively, heparin over a wide dynamic range of content could be detected. Typically, when JGB concentration is kept at 1.00 × 10⁻⁵ mol l⁻¹, using R_ratio could detect heparin over a range of 0.30–2000 ng ml⁻¹ with a limit of determination of 30 pg ml⁻¹, while using A_ratio could detect heparin over the range of 0.01–2000.0 ng ml⁻¹ with a limit of determination of 1 pg ml⁻¹. The wavelength-dependent RLS and absorption ratiometric methods were found to have much better flexibility for a series of common influences, such as pH, and dye concentration, than that of RLS method at a single wavelength. The interaction nature was investigated through size measurements and RLS imaging detections.

Introduction

Light scattering is a very common phenomenon and has been widely applied in size measurements and distributions of colloid and pharmaceutical particles [1], [2]. Resonance light scattering technique is a creative application of light scattering signals measured by simultaneously scanning the excitation and emission monochromators of a common spectrofluorometer [3], [4]. It has proved that RLS technique could be used for the assignments of bio-assemblies of drugs and biopolymers [3], [4], [5], [6]. For analytical purpose, RLS technique has the advantages of simplicity and high sensitivity for detecting biomolecules [7], [8], [9], [10], [11], nanoparticles [12], and drugs [13]. It has reported that the scattered light emission of gold particles is 10⁵-fold than the fluorescence emission of a fluorescein molecule [14], and could act as highly fluorescent analogs and tracer labels in clinical and biological applications [15], [16]. Thus, the RLS technique has shown high promise in analytical and biochemical sciences. In practical operation, however, the maximum RLS intensities at a single characteristic wavelength are not reliable to describe the properties for RLS spectra for their fluctuating variations at the single wavelength [17]. To overcome shortcomings of RLS method, herein we propose a RLS ratiometry measured using a common spectrofluorometer using the interaction between heparin and Janus Green Blue (JGB) as an example, and a comparison is made with the corresponding absorbance ratiometric method.

Ratiometric method could date from 1985 when Tsien and co-workers reported a ratio fluorescent indicator for selective calcium detection [17]. Ever since, wavelength-dependent ratiometric methods were reported in fluorescence domains as more effective analytical methods including fluorescence ratiometry and ratio imaging technique to analyze intracellular oxygen [18], pH [19], metal ions [20], aminophenol [21], DNA [22], and membrane potential [23], [24], [25], [26], [27], [28], [29] in living cells. These ratiometric methods have proved to hold the advantages over the conventional ones of single wavelength-intensity since they account for variations in experiments [30].

Heparin is a high negatively charged sugaramic polysaccharide belonging to amylose family. It has been widely applied to prevent thrombosis of the extracorporeal circuit and to mediate activation of the hemostatic system during surgery, acting as anticoagulant, atrology, anti-inflammation, anti-knub and virus demurrer [31]. Quantifying trace amount of heparin is very important, and it has been frequently carried out using spectrophotometric [32], chromatographic, electrophoretic [33], [34], and ion-selective electrode methods [35]. Our RLS ratiometric method of heparin, proposed herein based on the enhanced RLS signals resulting from its interaction with JGB through static attraction in neutral aqueous medium, is sensitive and reproducible compared to the common RLS method based on the measurements of scattered light intensities at a single wavelength.