A wide dynamic range detection of biopolymer medicines with resonance light scattering and absorption ratiometry
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.
Section snippets
Apparatus
The RLS spectra were obtained using a Hitachi F-2500 spectrofluorometer (Tokyo, Japan) by simultaneously scanning the excitation and emission monochromators without wavelength difference, while the absorption spectra were measured using a Hitachi 3010 spectrophotometer (Tokyo, Japan). A N5 PCS submicron particle size analyzer (Beckman Coulter, Miami, USA) was used to detect the size of micro-particles in solutions based on photon correlation spectroscopy (PCS). A home-developed laser-powered
spectroscopic characteristics of the interaction between heparin and JGB
Fig. 1 shows the RLS spectra of the interaction of heparin with JGB in weak basic medium. It could be seen that the RLS signals of both JGB and heparin are very weak. There is a characteristic RLS peak at 275 nm for heparin, and a characteristic RLS peak at 325 nm accompanied by a shoulder RLS peak at 280 nm for JGB. The weak RLS signals of JGB, however, could be greatly enhanced by heparin, showing a characteristic peak at 314 nm with several shoulder peaks in the region of 350.0–600.0 nm,
Conclusions
A dual wavelength ratiometric method herein is discussed and it provides a new generic approach, enlarging the applications of RLS and absorption methods. The analytical results of above data show that the advantages of the ratiometric methods seem obvious. At first, and the sensitivity and repeatability are much better than that of RLS method at a single wavelength. Secondly, not only does this dual-wavelength ratiometric method extend the linear relationship between the RLS and absorption
Acknowledgements
All authors herein are grateful to the supports from the National Natural Science Foundation of China (NSFC, No: 20425517, No: 20275032), the Program for New Century Excellent Talents in University (NCET-04-0852) and Chun Hui Program (No: [2004] 7-24) directed under the Ministry of Education of PRC, and the Municipal Science and Technology Committee of Chongqing.
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