请教纳米金标记凝血酶适配体的实验细节问题？

稳定纳米金的适配体的用量我已经确定了的，然后我用200uL的0.01%纳米金与一定量的29个碱基的凝血酶适配体混合以后，在25度温育了30分钟，再加入Tris-HCl 缓冲溶液和NaCl，然后加入一定量的凝血酶与之反应，再加入NaCl。但是却发现加入凝血酶的体系和没有加入凝血酶的体系都没有纳米金聚聚的现象，也就是没有变成紫色。继续增大加入的氯化钠浓度和凝血酶浓度（最高达到 0.5OD/mL）都没有看到聚集现象，反应均在25度下反应的。

对于这个现象，我有几个疑问：
1、不是因为适配体没有和金纳米结合好，导致凝血酶的加入没有使得纳米金聚集，那么适配体和纳米金结合的时间应该怎么控制？
2、是否因为凝血酶与适配体结合反应的时间和条件不对，导致凝血酶根本没有和适配体结合，那么这个时间和条件是什么？

3、缓冲液和电解质氯化钠、氯化钾是应该与适配体混合后加入纳米金；还是应该在纳米金和适配体结合以后再加进去缓冲溶液和电解质氯化钠和氯化钾？

想请教一下有做过这方面实验的朋友指点一下？


我是参考了下面一篇文章以及这篇文章的补充材料来做这个实验的。

[1]Simple and Sensitive Aptamer-based Colorimetric Sensing of Protein
Using Unmodified Gold Nanoparticle Probes[J].Hui Wei, Bingling Li, Jing Li, Erkang Wang and Shaojun Dong.Chem. Commun., 2007, 3735–3737 .（文献保存在网络下载地址中，可下载）

其中的主要实验步骤如下：

Preparation of Gold Nanoparticles (AuNPs). 13 nm AuNPs were prepared according to a literature method. Briefly, a sodium citrate solution (0.1 M, 1.94 mL) was rapidly added to a boiled HAuCl4 solution (50 mL H2O, 0.167 mL 10% HAuCl4) under vigorous stirring. The mixed solution was boiled for 10 min and further stirred for 15 min. The resulting wine-red solution was cooled to room temperature and filtered, which was stored in the 4 °C refrigerator before use.

Colorimetric Detection of Thrombin. A typical colorimetric analysis was realized as following procedure ：first, 200 μL 13 nm AuNPs was mixed with 30 μL 4.59 μM 29-mer thrombin binding aptamer (or control random oligonucleotide) in 20 mM Tris-HCl buffer containing 140 mM NaCl, 5 mM KCl (pH 7.5). Second, 30 μL thrombin with appropriate concentration (or BSA) in water was added to the AuNPs/aptamer solution. The solutions were allowed react for 5 min at room temperature and then 100 μL 0.5 M NaCl was added to produce color change. Time-dependent UVvisible spectra of the mixing AuNPs/aptamer/thrombin solution in the presence of NaCl were then measured with a Cary 500 Scan UV-Vis-NIR Spectrophotometer