Research

New type of chemical probe

           Chemical probes are important tools in basic biology research and medical diagnosis because they allow for sensitive, simple and specific detection of target molecules in complex environments, such as cell lysates, living cells, and in vivo. Currently, most of the chemical probes are reaction-based and designed for monitoring enzyme activities and reactive small molecules using fluorescence detection technique. Based on the fluorescence turn-on mechanism, they can be divided into two classes, dye-based fluorescent probe and caged-luciferin.

           Recently, our lab has developed a new type of chemical probe based on the controlled binding of streptavidin and biotin. The rationale behind this new concept is based on the fact that biotin has extremely high binding affinity (Kd = 10−14 M) with streptavidin, while chemical modification at N’-1 urea nitrogen of biotin to form caged-biotin can dramatically reduce its streptavidin binding affinity (Kd ≈ 10−5 M). In the absence of the target analyte, the caged-biotin probe on the cell surface would not be able to bind with fluorophore conjugated streptavidin due to the low binding affinity of caged-biotin with streptavidin. The fluorophore conjugatedstreptavidin can then be washed away to eliminate any background fluorescence. In the presence of the target analyte to trigger biotin uncaging, fluorophore conjugated streptavidin would bind to the biotin probe. As there are multiple fluorophore units on one streptavidin molecule, significant signal amplification can be achieved. Furthermore, streptavidin conjugated with different bright fluorophores, such as Cy5, Cy3 or Alexa488 can be added on demand to generate the desired fluorescent signals. Thus, the detection is no longer restricted by the type of fluorescent dyes, such as in the case of fluorescent probes.

Near-infrared fluorescent switchable dyes

           Our lab is also interested in developing new type of near-infrared (IR) fluorescent switchable dyes. Fluorescent dyes are a rapidly expanding area of research in chemical and biological sciences with multiple applications as biomolecule labels, enzyme substrates, environmental indicators and cellular stains. Fluorescent dyes that are excited and emit in the near-IR region are especially biocompatible and advantageous as they cause minimum damage to biological samples, have deep tissue penetration, and come under minimal interference from background autofluorescence by biomolecules in the living systems. Currently, most near-IR dyes are based on the cyanine and rhodamine scaffolds. Conventional strategies to obtain cyanine and rhodamine derivatives with appreciable bathochromic shift covering near-IR spectra include extending the p-conjugation and introducing heteroatoms and rigid bridges.

           Recently, we have introduced a novel near-IR fluorescence switchable merocyanine dye that can be coupled with different protein ligands to interact with non-enzymatic proteins for the rapid fluorescence turn-on labeling and imaging in living cells and in vivo.In contrast to the typical p-conjugation extension approach, our dye achieves bathochromic shift by the formation of an unusual S-cis diene conformer. Although bathochromic shifts due to S-cis conformation was first reported by Woodward several decades ago and are well documented in many literatures, the application of this conformation in fluorescent dyes has not been demonstrated. This is the first time that a stable S-cis conformation has been successfully identified in a near-IR dye. In addition to the novel bathochromic shift mechanism, our new dye also exhibits fluorescence-switchable properties in response to polarity and viscosity. When different protein specific ligands were conjugated to the dye, the probes show a dramatic increase in fluorescence (up to 300-fold) in the presence of target proteins (SNAP-tag and hCAII proteins).

新穎化學探針

小分子化學螢光探針為基礎生物學研究與醫學診斷中一相當重要的工具。基於螢光開啟機制，它們可以被分成兩種，螢光染料探針(dye-based fluorescent probe)和籠閉型螢光素(caged-luciferin)。目前，多數的螢光探針被應用於各種酵素活性與反應性小分子的偵測。它們能對目標分析物進行靈敏、簡易且專一的檢測，因此被廣泛應用在複雜的生物樣品中，如細胞裂解液、活細胞與動物體內。

近年來，我們開發出一種新型的化學螢光探針，其基本原理是生物素跟鏈黴親合素具有高親和性的結合(Kd = 10-14 M)，然而在生物素的1號或3號氮上進行化學修飾形成籠閉生物素，可顯著降低其與鏈黴親合素的親和力(Kd = 10-5 M)。因此，運用鏈黴親合素與生物素和籠閉生物素間巨大的親和力差異，可達到螢光訊號的可控開啟。

在目標分析物不存在的情況下，由於籠閉生物素與鏈黴親合素間太低的親和力，使得籠閉生物素探針不能與已修飾上螢光團的鏈黴親合素結合，經清洗后可消除螢光背景。在目標分析物存在下觸發籠閉生物素解除籠閉，使得已修飾上螢光團的鏈黴親合素可以和生物素探針進行結合，產生螢光訊號。由於一個鏈黴親合素上含有多個螢光團，此方法可達到訊號放大的效果。此外，依據實驗需求，可以修飾上不同的螢光團，例如:Cy5、Cy3或是Alexa488以產生不一樣波長的螢光訊號，因此，偵測上不再受到螢光染料的限制。

近紅外光可切換螢光染料

我們實驗室也致力於開發新型近紅外螢光染料。螢光染料在化學與生物科學領域快速發展，其用途廣泛，可被應用於生物分子標記、酵素探針、環境指示劑與細胞染色劑等。其中， 在近紅外區激發與發射的螢光染料具有高生物相容性，因其可減少生物樣品破壞、深度組織滲透與降低生物分子自體螢光背景干擾等優點。目前，大多數近紅外光染料是以花青素與羅丹明為基礎。產生近紅外光花青素與羅丹明衍生物的策略包含延長p-共軛、引入雜原子（heteroatoms）與剛性橋樑 (rigid bridges)。

最近，我們合成了一種新型的近紅外可切換螢光花青染料，相較於典型的p-共軛延伸，此新染料藉由異常的S-順式二烯構型(S-cis conformation)來達到螢光紅移的效果。S-順式構型的紅移現象，在數十年前首先由Woodward教授所報導，在目前的許多教科書與文獻也有詳細的記載，但此構型從未在螢光染料中得到證實及應用，我們的實驗室第一次合成出穩定的S-順式構造的近紅外光染料。除了新穎的紅移機制外，此染料還具有在高極性與黏稠性環境中螢光增强的特性。當不同的蛋白質配體(SNAP-tag 與hCAII蛋白)與此類染料結合時，探針在目標蛋白存在下會有明顯的螢光上升(高達300倍)。因此，此類染料可以被應用於快速蛋白偵測、細胞與動物活體影像。