A resonance energy transfer between chemiluminescent donors and luminescent quantum-dots as acceptors (CRET)
A resonance energy transfer between chemiluminescent donors and luminescent quantum-dots as acceptors (CRET)
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DOI：
10.1002/anie.200601196
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发表时间：
2006-01-01
影响因子：
16.6
通讯作者：
Ren, Jicun
中科院分区：
文献类型：
--
作者：
Huang, Xiangyi;Li, Liang;Ren, Jicun
Scheme1。在Scheme1A中，CL给体luminol没有直接与量子点相连，而催化剂HRP则与量子点共轭。HRP能连续催化鲁米诺/过氧化氢CL反应。在该系统中，QD-HRP偶联物可以用作细胞和组织成像的探针，类似于BRET方案1B中，QDs与牛血清白蛋白（BSA）结合，HRP与牛血清白蛋白抗体（抗BSA）结合。当抗bsa - hrp与BSA-QDs结合时，可发生CRET。该系统有潜力用于非竞争和竞争模式的免疫测定（见支持信息）。本研究以巯基丙酸（MPA）为稳定剂，在水相中以Cd2+与NaHTe溶液反应合成了不同尺寸的水溶性CdTe量子点，其qy值为40-50%。利用EDC（1-乙基-3-（3-二甲氨基丙基）碳二亚胺）作为偶联剂，将mpa包被的CdTe量子点与某些蛋白质（如HRP和BSA）偶联。用超滤膜对混合物进行纯化。用激光诱导荧光检测器对QD生物偶联物进行了毛细管电泳表征。结果表明，超滤纯化QD-HRP偶联物是一种简单有效的方法，偶联物保存一周后保持稳定（见支持资料）。利用球面近似法估计HRP的直径（MW= 40kDa）为35通过荧光相关光谱（FCS）测定622QD - hrp偶联物和622QD（即发射波长为622 nm的量子点）的水动力直径[23]分别约为8 nm和4 nm（见支持资料）。每个622QD-HRP偶联物估计含有一个或两个固定的HRP拷贝。我们研究了QD - hrp系统中鲁米诺和QD生物偶联物之间的CRET，结果如图2所示。在图2A中，四个不同尺寸的量子点（发射波长分别为557、587、622和657nm）被用作受体。在鲁米诺和QD生物偶联物之间观察到一个有效的CRET，看起来像FRET。CRET比率由受体发射率除以供体发射率确定，范围为0.22至0.32。不同的量子点之间的CRET效率差异较小，主要归因于它们不同的供体-受体距离和量子产率。
Scheme1. In Scheme1A, the CL donor, luminol, is not directly linked with the QDs, and the catalyst, HRP, is conjugated to the QDs. HRP can continuously catalyze the luminol/hydrogen peroxide CL reaction. In this system, the QD–HRP conjugates can be used as probes in cell and tissue imaging similar to BRET.[14] In Scheme 1B, QDs are linked with bovine serum albumin (BSA), and HRP is conjugated with the BSA antibody (anti-BSA). When the anti-BSA–HRP binds to the BSA–QDs, CRET can occur. This system has potential to be used in immunoassay in non-competition and competition modes (see Supporting Information). In our study, different sized water-soluble CdTe QDs were synthesized in the aqueous phase using the reaction between Cd2+ and NaHTe solution in the presence of mercaptopropyl acid (MPA) as a stabilizer [21] and their QYs were measured to be 40–50%. The MPA-coated CdTe QDs were conveniently conjugated to certain proteins (such as HRP and BSA) using EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide) as a coupling reagent. The mixtures were purified using ultrafiltration membrane. The QD bioconjugates were characterized by capillary electrophoresis with laser-induced fluorescent detector. Our results showed that ultra-filtration was an effective and simple approach to purify QD–HRP conjugates and the conjugates were stable after being kept for one week (see Supporting Information). The diameter of HRP (MW= 40kDa) was estimated to be 35, by using spherical approximation.[22] The hydrodynamic diameter of 622QD–HRP conjugates and 622QD (that is, QDs with an emission wave length of 622 nm), measured by fluorescence correlation spectroscopy (FCS),[23] were approximately 8 nm and 4 nm, respectively (see Supporting Information). Each 622QD–HRP conjugate was estimated to contain one or two copies of immobilized HRP.We investigated the CRET between luminol and QD bioconjugates in the QD–HRP system and the results obtained are shown in Figure 2. In Figure 2A, four different sized QDs (with emissions at 557, 587, 622, and 657 nm) were used as acceptors. An efficient CRET between luminol and QD bioconjugate was observed, which looked like FRET. The CRET ratios, determined by dividing the acceptor emission by the donor emission, ranged from 0.22 to 0.32. The small differences of the CRET efficiency among the QDs are attributed to their different donor–accepter distances and quantum yields.