van der Sneppen L, Buijs J B, Gooijer C, Ubachs W, Ariese F
Laser Centre, Vrije Universiteit, De Boelelaan 1081-1083, 1081 HV Amsterdam, The Netherlands.
Appl Spectrosc. 2008 Jun;62(6):649-54. doi: 10.1366/000370208784657977.
The feasibility of liquid-phase evanescent-wave cavity ring-down spectroscopy (EW-CRDS) for surface-binding studies under flow-injection analysis (FIA) conditions is demonstrated. The EW-CRDS setup consists of an anti-reflection coated Dove prism inside a linear cavity (with standard or super-polishing of the total internal reflective (TIR) surface). A teflon spacer with an elliptical hole clamped on this surface acts as a 20 muL sized flow cell. The baseline noise of this system is of the order of 10(-4) absorbance units; the baseline remains stable over a prolonged time and the prism surface does not become contaminated during repeated injections of the reversibly adsorbing test dyes Crystal Violet (CV) and Direct Red 10 (DR10). At typical FIA or liquid chromatography (LC) flow rates, the system has sufficient specificity to discriminate between species with different surface affinities. For CV a much stronger decrease in ring-down time is observed than calculated based on its bulk concentration and the effective depth probed by the evanescent wave, indicating binding of this positively charged dye to the negatively charged prism surface. The amount of adsorption can be influenced by adjusting the flow rate or the eluent composition. At a flow rate of 0.5 mL/min, an enrichment factor of 60 was calculated for CV; for the poorly adsorbing dye DR10 it is 5. Super-polishing of the already polished TIR surface works counter-productively. The adsorbing dye Crystal Violet has a detection limit of 3 muM for the standard polished surface; less binding occurs on the super-polished surface and the detection limit is 5 muM. Possible applications of EW-CRDS for studying surface binding or the development of bio-assays are discussed.
证明了液相倏逝波腔衰荡光谱法（EW-CRDS）在流动注射分析（FIA）条件下用于表面结合研究的可行性。EW-CRDS装置由线性腔内的抗反射涂层道威棱镜组成（全内反射（TIR）表面采用标准或超抛光处理）。一个带有椭圆孔的聚四氟乙烯垫片夹在该表面上，用作20 μL大小的流动池。该系统的基线噪声约为10^(-4)吸光度单位；基线在较长时间内保持稳定，并且在反复注入可逆吸附的测试染料结晶紫（CV）和直接红10（DR10）期间，棱镜表面不会被污染。在典型的FIA或液相色谱（LC）流速下，该系统具有足够的特异性，能够区分具有不同表面亲和力的物质。对于CV，观察到的衰荡时间下降比根据其本体浓度和倏逝波探测的有效深度计算得出的要强得多，这表明这种带正电的染料与带负电的棱镜表面发生了结合。吸附量可以通过调节流速或洗脱液组成来影响。在流速为0.5 mL/min时，计算出CV的富集因子为60；对于吸附性较差的染料DR10，富集因子为5。对已经抛光的TIR表面进行超抛光起到了适得其反的作用。对于标准抛光表面，吸附染料结晶紫的检测限为3 μM；在超抛光表面上结合较少，检测限为5 μM。讨论了EW-CRDS在研究表面结合或生物分析开发方面的可能应用。
