Chemistry Department, University of Rome , Tor Vergata, Via della Ricerca Scientifica, 00133, Rome, Italy.
Département de Chimie, Université de Montréal , Montréal, Québec H3C 3J7, Canada.
Acc Chem Res. 2016 Sep 20;49(9):1884-92. doi: 10.1021/acs.accounts.6b00276. Epub 2016 Aug 26.
The biosensor community has long focused on achieving the lowest possible detection limits, with specificity (the ability to differentiate between closely similar target molecules) and sensitivity (the ability to differentiate between closely similar target concentrations) largely being relegated to secondary considerations and solved by the inclusion of cumbersome washing and dilution steps or via careful control experimental conditions. Nature, in contrast, cannot afford the luxury of washing and dilution steps, nor can she arbitrarily change the conditions (temperature, pH, ionic strength) under which binding occurs in the homeostatically maintained environment within the cell. This forces evolution to focus at least as much effort on achieving optimal sensitivity and specificity as on achieving low detection limits, leading to the "invention" of a number of mechanisms, such as allostery and cooperativity, by which the useful dynamic range of receptors can be tuned, extended, narrowed, or otherwise optimized by design, rather than by sample manipulation. As the use of biomolecular receptors in artificial technologies matures (i.e., moves away from multistep, laboratory-bound processes and toward, for example, systems supporting continuous in vivo measurement) and these technologies begin to mimic the reagentless single-step convenience of naturally occurring chemoperception systems, the ability to artificially design receptors of enhanced sensitivity and specificity will likely also grow in importance. Thus motivated, we have begun to explore the adaptation of nature's solutions to these problems to the biomolecular receptors often employed in artificial biotechnologies. Using the population-shift mechanism, for example, we have generated nested sets of receptors and allosteric inhibitors that greatly expanded the normally limited (less than 100-fold) useful dynamic range of unmodified molecular and aptamer beacons, enabling the single-step (e.g., dilution-free) measurement of target concentrations across up to 6 orders of magnitude. Using this same approach to rationally introduce sequestration or cooperativity into these receptors, we have likewise narrowed their dynamic range to as little as 1.5-fold, vastly improving the sensitivity with which they respond to small changes in the concentration of their target ligands. Given the ease with which we have been able to introduce these mechanisms into a wide range of DNA-based receptors and the rapidity with which the field of biomolecular design is maturing, we are optimistic that the use of these and similar naturally occurring regulatory mechanisms will provide viable solutions to a range of increasingly important analytical problems.
生物传感器领域长期以来一直致力于实现尽可能低的检测极限，而特异性（区分紧密相似的靶分子的能力）和灵敏度（区分紧密相似的靶浓度的能力）在很大程度上被视为次要考虑因素，并通过繁琐的洗涤和稀释步骤或通过仔细控制实验条件来解决。相比之下，自然界无法承受洗涤和稀释步骤的奢侈，也不能随意改变细胞内维持的内环境中结合发生的条件（温度、pH 值、离子强度）。这迫使进化至少在实现最佳灵敏度和特异性方面投入同样多的努力，以实现低检测极限，从而导致了许多机制的“发明”，例如变构和协同作用，通过这些机制可以调整、扩展、缩小或通过设计优化受体的有用动态范围，而不是通过样品处理。随着生物分子受体在人工技术中的应用不断成熟（即，从多步、实验室绑定的过程转移到例如支持连续体内测量的系统），并且这些技术开始模拟自然发生的化学感受系统的无试剂单步便利性，增强灵敏度和特异性的人工设计受体的能力也可能变得更加重要。出于这个动机，我们已经开始探索将自然界的解决方案应用于这些问题，以适应人工生物技术中常用的生物分子受体。例如，我们使用种群转移机制生成嵌套受体和变构抑制剂，极大地扩展了未经修饰的分子和适体信标的通常有限的（小于 100 倍）有用动态范围，从而能够在多达 6 个数量级上进行目标浓度的单步（例如，无需稀释）测量。通过使用相同的方法将隔离或协同作用合理地引入这些受体中，我们同样将其动态范围缩小到 1.5 倍，极大地提高了它们对靶配体浓度微小变化的响应灵敏度。鉴于我们能够轻松地将这些机制引入广泛的基于 DNA 的受体中，并且生物分子设计领域的发展速度非常快，我们乐观地认为，这些和类似的自然发生的调节机制将为一系列越来越重要的分析问题提供可行的解决方案。
