As masters of molecular recognition, proteins are key components in many molecular sensors, either as part of a sensor device or as a stand-alone sensor. The former includes proteins involved in molecular recognition (antibodies, ligand binding domains, DNA-binding proteins, enzymes) or in signal generation (reporter enzymes, fluorescent proteins, polymerases). Molecular recognition and signal generation are integrated in a single sensor protein in stand-alone sensors, rendering these sensors attractive as genetically encoded sensors for intracellular sensing, and as sensor proteins for point-of-care diagnostics. Despite its importance, the role of protein engineering in sensor development remains sometimes underappreciated, and many opportunities remain for protein engineering and synthetic biology to contribute to the sensor field. In this Virtual Issue (https://pubs.acs.org/page/ascefj/vi/protein-engineering-sensors), we have selected 27 recent articles from ACS Sensors, ACS Chemical Biology, Bioconjugate Chemistry, and ACS Synthetic Biology that represent the most recent developments in the engineering of new proteins for sensing, and together illustrate the importance of protein engineering to the field of sensing. A very active area of protein sensor engineering is the development of genetically encoded fluorescent sensors that can be used for intracellular live-cell imaging. Two basic types of sensors have been developed in this area: those depending on the modulation of fluorescence of a single fluorescent protein domain (often a circularly permuted fluorescent protein), and those depending on the modulation of Förster Resonance Energy Transfer (FRET) between a donor and an acceptor domain. Whereas most work in this area focuses on sensor development for a specific application, several groups also aim to develop general platforms that allow the rational design of sensor proteins, develop efficient library screening methods, or provide more insight into the working mechanism of existing sensor proteins. An interesting example of the latter is the work of Höfig and co-workers, who used single-molecule fluorescent detection to provide in-depth insight into the conformational dynamics of two previously developed FRET sensor proteins for glucose and crowding.(1) Wu and co-workers developed a red variant of a genetically encoded glutamate sensor protein by replacing the green fluorescent domain in a previously developed glutamate sensor (iGluSnFr) by a circularly permuted red fluorescent protein, and subsequently used directed evolution to identify linker-variants that restored optimal allosteric coupling between the glutamate receptor domain and the fluorescent reporter.(2) The newly developed red fluorescent glutamate sensors were successfully used to resolve glutamate transients in electrically stimulated hippocampal neurons. Single domain fluorescent sensors typically display a large dynamic range, but have the drawback of being intensiometric, making them less suited for quantitative measurements. Several groups have addressed this drawback by turning intensiometric sensors into ratiometric sensors. Burgstaller et al. reported the development of pH-Lemon by fusing the bright pH-stable cyan fluorescent protein mTurqoise2 to the intrinsically pH-sensitive yellow fluorescent protein EYFP.(3) The FRET-based pH-lemon sensor proved particularly useful for accurate quantification of pH in acidic vesicles, using donor lifetime imaging. Another approach to render an intensiometric sensor into a ratiometric sensor is to append an analyte insensitive fluorescent domain to allow for internal calibration. Fudge and co-workers first increased the intensiometric response of their green Zn²⁺ sensor GZnP1 2-fold by improving the allosteric coupling between the Zn²⁺ binding zinc finger domain and circularly permuted GFP by directed evolution of linker regions.(4) Next, fusion of a Zn²⁺-insensitive red fluorescent protein (mCherry) allowed quantitation of free Zn²⁺ levels in both mitochondrial matrix and the mitochondrial intermembrane space using excitation ratiometric imaging. Whereas GZnP1 was designed such that very little, or no, energy transfer occurred between the green and red fluorescent domains, Norcross and co-workers developed red variants of redox-sensitive GFP (roGFP) by making a tight fusion of roGFP with a variety of red fluorescent protein acceptors.(5) The availability of these red-light emitting redox-sensitive sensor proteins allows simultaneous redox state monitoring in different intracellular locations, using red-emitting sensor proteins targeted to the mitochondria while expressing the original green emitting roGFP in the cytosol, for example. Unlike many small-molecule reaction-based fluorescent probes, these genetically encoded sensor proteins are reversible, allowing them to monitor the redox status in real time. Several groups have also explored the benefits of using semisynthetic fluorescent sensor proteins, as discussed in a timely review by Ueda et al.(6) These sensors allow the use of environmentally sensitive synthetic fluorophores via in situ noncovalent self-assembly, metal-mediated coordination, or the use of self-labeling protein domains such as Halo- and SNAP tags. In work published in ACS Sensors, Johnsson and co-workers reported the development and application of semisynthetic FRET sensors to monitor drug uptake in living cells.(7) In these SNIFIT sensors the target protein of a given drug is used as a receptor module to generate a FRET-based protein switch. Binding of the drug molecule competes with an intramolecular interaction that keeps two fluorescent domains in close proximity, providing a broadly applicable and robust sensor mechanism. By allowing real-time monitoring of cellular drug uptake and/or membrane permeability this technology may become an efficient tool to screen for drug efficacy in an early stage of drug development. Another example of semisynthetic fluorescent sensors is the new biosensor design developed by Tebo and co-workers based on circularly permuted variants of the Fluorescence-Activating and absorption-Shifting Tag (cpFAST).(8) FAST domains are derivatives of photoactive yellow proteins that bind and increase the fluorescence of various hydroxybenzylidene rhodamine (HBR derivatives). Binding of Ca²⁺ to a calmodulin/M13 pair fused to cpFAST increased the affinity of the FAST domain for HBR, resulting in a Ca²⁺-dependent increase in fluorescence intensity. Unlike fluorescence, whose dependence on external illumination gives rise to autofluorescence and scattering, bioluminescence is better suited for in vivo imaging and for measuring analytes directly in complex media such as blood. In addition, bioluminescent sensor proteins are attractive for light-sensitive applications, including optogenetics and long-term continuous monitoring. A popular approach to generate bioluminescent sensor proteins is to use control of intramolecular complementation of a split luciferase. Hossain and co-workers used the well-known Ca²⁺-induced interaction between calmodulin and M13 to drive the intramolecular complementation of split NanoLuc luciferase.(9) Tight fusion of this sensor part to cyan, green, and yellow fluorescent domains yielded three bioluminescent color variants by energy transfer from the luciferase to the fluorescent domain. A similar approach was pursued by Dippel et al. to develop a bioluminescent sensor protein for the bacterial second messenger cyclic di-GMP.(10) Screening a small library of 92 variants yielded sensor proteins with an impressive 90-fold increase in bioluminescent signal and a physiologically relevant affinity. Whereas these sensors allowed only bioluminescent detection, Farhana and co-workers recently reported the development of new genetically encoded Ca²⁺ indicator (GECI) that allows both fluorescent and bioluminescent detection.(11) The reported GLICO sensor used the Ca²⁺-induced interaction between calmodulin and M13 to simultaneously drive the intramolecular complementation of a split luciferase and restore fluorescence in a circularly permuted GFP domain. A new class of ratiometric bioluminescent sensor proteins based on intramolecular complementation of split NanoLuc luciferase was reported by Ni and co-workers.(12) In their antibody sensors, a single copy of a large split luciferase domain was fused via flexible linkers to two copies of the complementary small split luciferase domain, of which one was labeled with a fluorescent acceptor. In the absence of the antibody, the fluorescently labeled small domain would bind to the large domain, giving rise to efficient BRET and red emission. Antibody binding to the sensor protein disrupted this interaction, allowing complex formation with the nonlabeled small luciferase domain, resulting in the native blue Nanoluc emission. In addition to applying (split) luciferases for various sensing applications, luciferase properties such as color, brightness, and protein stability continue to be improved. Gaur and co-workers re-engineered Gaussia luciferase, a very bright and stable luciferase that is normally secreted from cells, to be retained in cells while still being efficiently folded.(13) Gaussia luciferase has thus become a viable alternative luciferase for intracellular sensing and imaging applications, which was demonstrated by developing it into a sensor for ER stress and caspase activation. Coelenterazine-using marine luciferases such as NanoLuc and Gaussia luciferases typically emit blue light, which is less attractive for in vivo applications. The group of Ai recently reported the development of a series of pyridyl-functionalized substrate variants with red-shifted emission and used directed evolution to generate LumiLuc luciferase variants with a broad substrate specificity that can accommodate these substrates.(14) While the importance of protein engineering is clearly visible in the field of bioluminescent and fluorescent sensor proteins, protein engineering also plays a key role in many other sensor formats. O’Neill and Lauglin reported the construction of a genetically encoded recording system for neuronal Ca²⁺ spikes based on the Ca²⁺-triggered reconstitution of a split protease system.(15) Protease activity was monitored by cleavage of a caged optical reporter, but the modularity of this system also allows it to be connected to other downstream processes. Several types of biological nanopore proteins have been successfully used for next-generation DNA sequencing and other sensing applications. Work by the group of Long has shown that well-chosen single point mutations of charged residues at the entrance or the lumen of the aerolysine channel protein could enhance both the sensitivity and selectivity of aerolysine-biosensors.(16) More drastic re-engineering of natural channel proteins was reported by Liu and co-workers, who aimed to further increase the channel diameter of the β-barrel protein ferric hydroxymate uptake protein component A (FhuA) by increasing the number of β-strands from 22 in the wt protein up to 34.(17) An increase in pore size was indeed observed up to a total of 30 β-strands, whereas insertion of additional β-strands resulted in the collapse of the pore and a smaller effective channel diameter. Many studies utilizing biosensors have been published in ACS Synthetic Biology. For example, bacterial transcription factors play essential roles in the engineering of complex biological circuits. Using molecular docking studies with sensors and site-directed mutagenesis, Silva-Rocha et al. engineered new transcription factors with enhanced responses to certain molecules, like benzoate and salicylic acid, for eliciting gene expression in E. coli.(18) In another study, Fields and colleagues recently reported on the generation of Saccharomyces cerevisiae digoxigenin and progesterone biosensors based on destabilized dimeric ligand-binding domains that undergo ligand-induced stabilization.(19) These biosensors will increase the flexibility for the construction of logic gates and other potential applications in the future. In another study, Alexandrov and colleagues used SpyTag/SpyCatcher-mediated protein ligation to engineer modularly organized, scaffold-dependent protease sensors to understand protease-inducible protein–protein interactions better.(20) In this way a suite of integrated signal sensing and amplification circuits was created that can detect the activity of α-thrombin and prostate-specific antigen. Biological sensors also play key roles in optogenetics, a field where researchers combine light and genetically engineered photoreceptors to precisely control certain biological processes. In a recent article, Tabor et al. engineered a novel E. coli near-infrared light sensor.(21) Unlike other systems in the literature, their new system did not require any secondary messengers, thus simplifying the process and allowing for rapid response dynamics. In another optogenetics study, Weber and colleagues engineered a system based on the light-sensitive bacterial transcription factor CarH and its cognate DNA operator sequence CarO from T. thermophilus to control gene expression in mammalian cells.(22) The construction of sensors also relies on the chemical construction of bioconjugates, and several articles in Bioconjugate Chemistry have described this type of work. One important use of biological sensors is the study of proteostasis, which is the process of cellular stress disrupting the homeostasis of proteins. Fares et al. developed a fluorogenic proteome stress sensor that could be used to monitor protein folding and provide insight into how cellular stress can impact the proteome.(23) When conjugated to standard immunoglobulins, Ackerman et al. showed that fluorogen-activating proteins could be used for antigen detection and cell ablation in vitro.(24) There have been some limitations in the development of specific modulators that can be used for high throughput screening technologies. For example, Sakyiamah and colleagues designed a novel NanoBRET-based sensor that could be used to screen for CXCR4 ligands.(25) As CXCR4 plays roles in many pathogens, such as HIV, this improved method may lead to new CXCR4-targeted drugs in the future. In another study, Ahmed et al. developed a FRET-sensor to visualize the delivery and release of small RNAs.(26) Their findings suggest that small RNA delivery vehicles may be useful to better understand noncoding RNA biology. Next, as a new type of luciferase, NanoLuc is 100-times brighter than traditional luciferases and, due to its small size, has minimal effect on protein function. In a recent study, Du et al. designed a genetically encoded biosensor, FapR-Nluc, which was created by conjugating NLuc with a malonyl-CoA response bacterial transcription factor.(27) The new biosensor allowed for the detection of malonyl-CoA levels in vitro and may be translated to mice in the future. The articles selected in this Virtual Issue represent persuasive examples of how protein engineering and synthetic biology can contribute to the sensor field. The engineering of proteins has enabled many breakthroughs in biosensing with improved functionality, and we are convinced that protein engineering will continue to play a crucial role in the development of new biosensor technology. Views expressed in this editorial are those of the authors and not necessarily the views of the ACS. This article references 27 other publications.

中文翻译：

---

工程传感器蛋白。

作为分子识别的大师，蛋白质是许多分子传感器的重要组成部分，它们既可以作为传感器设备的一部分，也可以作为独立传感器使用。前者包括与分子识别（抗体，配体结合域，DNA结合蛋白，酶）或信号生成（报告酶，荧光蛋白，聚合酶）有关的蛋白质。分子识别和信号生成集成在独立传感器中的单个传感器蛋白质中，使这些传感器作为用于细胞内传感的基因编码传感器以及作为即时诊断的传感器蛋白质具有吸引力。尽管具有重要意义，但蛋白质工程在传感器开发中的作用有时仍未被重视，并且蛋白质工程和合成生物学在传感器领域做出贡献的机会仍然很多。ACS传感器，ACS化学生物学，生物共轭化学和ACS合成生物学这些代表了传感新蛋白工程领域的最新进展，并共同说明了蛋白质工程对传感领域的重要性。蛋白质传感器工程的一个非常活跃的领域是开发可用于细胞内活细胞成像的遗传编码荧光传感器。在这一领域已开发出两种基本类型的传感器：一种取决于单个荧光蛋白结构域（通常是环形排列的荧光蛋白）的荧光调制，另一种取决于传感器之间的Förster共振能量转移（FRET）的调制。供体和受体域。尽管该领域的大部分工作都集中在针对特定应用的传感器开发上，但也有几个小组的目标是开发允许合理设计传感器蛋白的通用平台，开发有效的文库筛选方法，或提供对现有传感器蛋白工作机制的更多见解。后者的一个有趣的例子是Höfig及其同事的工作，他们使用单分子荧光检测技术深入了解了两个先前开发的FRET传感器蛋白质在葡萄糖和拥挤方面的构象动力学。（1）Wu和同事通过用圆形排列的红色荧光蛋白替换先前开发的谷氨酸传感器（iGluSnFr）中的绿色荧光结构域，开发了遗传编码的谷氨酸传感器蛋白的红色变体，随后利用定向进化来鉴定恢复了最佳状态的接头变体谷氨酸受体结构域与荧光报告分子之间的变构偶联。（2）新开发的红色荧光谷氨酸传感器成功地用于解决电刺激海马神经元中的谷氨酸瞬变。单域荧光传感器通常显示较大的动态范围，但具有强度测量的缺点，使其不适用于定量测量。几个小组通过将强度传感器变成比例传感器来解决这个缺点。Burgstaller等。报道了通过将明亮的pH稳定的蓝绿色荧光蛋白mTurqoise2与本质上对pH敏感的黄色荧光蛋白EYFP融合而开发pH柠檬的技术。（3）基于FRET的pH柠檬传感器特别适用于对酸性pH的精确定量。囊泡，使用供体寿命成像。将强度传感器变成比例传感器的另一种方法是将分析物不敏感的荧光域附加到内部进行校准。福吉和他的同事们首先提高了他们绿色锌的强度测量响应²⁺传感器GZnP1通过连接子区域的定向进化改善Zn ²⁺结合的锌指结构域与圆形排列的GFP之间的变构偶联而提高了2倍。（4）接下来，融合Zn ^2+不敏感的红色荧光蛋白（mCherry） ）允许定量游离Zn ²⁺激发比率成像法检测线粒体基质和线粒体膜间空间的水平。设计GZnP1时，绿色和红色荧光域之间几乎没有或没有发生能量转移，而Norcross和同事通过将roGFP与多种荧光蛋白紧密融合，开发出了氧化还原敏感的GFP（roGFP）的红色变体。红色荧光蛋白受体。（5）这些发红光的氧化还原敏感传感器蛋白的可用性允许使用针对线粒体的发红光传感器蛋白在细胞内的不同位置同时进行氧化还原状态监测，同时在细胞中表达原始发绿光的roGFP。例如，胞质溶胶。与许多基于小分子反应的荧光探针不同，这些遗传编码的传感器蛋白是可逆的，允许他们实时监控氧化还原状态。如Ueda等人（6）的及时评论所讨论的，几个小组还探索了使用半合成荧光传感器蛋白的好处。这些传感器允许通过以下方式使用对环境敏感的合成荧光团：原位非共价自组装，金属介导的配位或使用自我标记蛋白结构域（例如Halo和SNAP标签）。在ACS传感器中发表的工作中，Johnsson及其同事报告了半合成FRET传感器的开发和应用，以监测活细胞中的药物吸收。（7）在这些SNIFIT传感器中，给定药物的靶蛋白被用作受体模块，以生成基于FRET的蛋白转变。药物分子的结合与分子内相互作用竞争，该相互作用使两个荧光结构域保持紧密接近，从而提供了广泛适用且强大的传感器机制。通过允许实时监测细胞药物摄取和/或膜通透性，该技术可以成为在药物开发早期筛选药物功效的有效工具。半合成荧光传感器的另一个示例是Tebo和同事基于循环排列的荧光激活和吸收移位标签（cpFAST）变体开发的新型生物传感器设计。（8）FAST域是结合了光敏黄色蛋白的衍生物并增加各种羟基亚苄基罗丹明（HBR衍生物）的荧光。Ca的结合²⁺到钙调蛋白/ M13对稠合到cpFAST增加了FAST域的亲和力HBR，产生的Ca ²⁺荧光强度依赖性增加。与依赖外部照明的荧光会引起自发荧光和散射不同，生物发光更适合于体内成像以及直接在复杂介质（例如血液）中测量分析物。此外，生物发光传感器蛋白对于光敏应用（包括光遗传学和长期连续监测）具有吸引力。产生生物发光传感器蛋白的流行方法是使用分裂荧光素酶的分子内互补控制。侯赛因及其同事使用了著名的Ca ²⁺介导的钙调蛋白与M13相互作用，驱动分裂的NanoLuc荧光素酶的分子内互补。（9）通过将能量从荧光素酶转移至荧光域，使该传感器部分与青色，绿色和黄色荧光域紧密融合，产生了三种生物发光颜色变体。 。Dippel等人也采用了类似的方法。以开发用于细菌第二信使环di-GMP的生物发光传感器蛋白。（10）筛选一个由92个变体组成的小文库，可产生具有显着90倍生物发光信号增强和生理相关亲和力的传感器蛋白。这些传感器仅允许生物发光检测，而Farhana及其同事最近报告了新的遗传编码的Ca ^2+的开发指示剂（GECI），可同时进行荧光和生物发光检测。（11）报告的GLICO传感器使用Ca ²⁺诱导的钙调蛋白与M13之间的相互作用，以同时驱动分裂的萤光素酶的分子内互补并在圆形排列的GFP域中恢复荧光。Ni和他的同事报道了一种基于分子内互补的NanoLuc荧光素酶的新型比例生物发光传感器蛋白。（12）在他们的抗体传感器中，一个大的拆分荧光素酶结构域的单个拷贝通过柔性接头融合到了两个拷贝上。互补小分裂荧光素酶结构域的一部分，其中一个被荧光受体标记。在没有抗体的情况下，荧光标记的小结构域将与大结构域结合，从而产生有效的BRET和红色发射。抗体与传感器蛋白的结合破坏了这种相互作用，允许与未标记的小荧光素酶结构域形成复合物，从而导致天然的蓝色Nanoluc发射。除了将（拆分的）萤光素酶应用于各种传感应用之外，萤光素酶的性质，例如颜色，亮度和蛋白质稳定性也不断得到改善。Gaur和他的同事重新设计了高斯荧光素酶，一种通常从细胞中分泌出来的非常明亮且稳定的荧光素酶，可以保留在细胞中，同时仍然可以有效折叠。（13）高斯荧光素酶因此成为一种可行的替代荧光素酶，用于细胞内感测。和成像应用，通过将其开发成用于ER应力和caspase激活的传感器得到了证明。使用腔肠素的海洋荧光素酶（例如NanoLuc和Gaussia荧光素酶）通常会发出蓝光，对于 导致天然的蓝色Nanoluc发射。除了将（拆分的）萤光素酶应用于各种传感应用之外，萤光素酶的性质，例如颜色，亮度和蛋白质稳定性也不断得到改善。Gaur和他的同事重新设计了高斯荧光素酶，一种通常从细胞中分泌出来的非常明亮且稳定的荧光素酶，可以保留在细胞中，同时仍然可以有效折叠。（13）高斯荧光素酶因此成为一种可行的替代荧光素酶，用于细胞内感测。和成像应用，通过将其开发成用于ER应力和caspase激活的传感器得到了证明。使用腔肠素的海洋荧光素酶（例如NanoLuc和Gaussia荧光素酶）通常会发出蓝光，对于 导致天然的蓝色Nanoluc发射。除了将（拆分的）萤光素酶应用于各种传感应用之外，萤光素酶的特性（例如颜色，亮度和蛋白质稳定性）继续得到改善。Gaur和他的同事重新设计了高斯荧光素酶，一种通常从细胞中分泌出来的非常明亮且稳定的荧光素酶，可以保留在细胞中，同时仍然可以有效折叠。（13）高斯荧光素酶因此成为一种可行的替代荧光素酶，用于细胞内感测。和成像应用，将其开发成用于内质网应激和胱天蛋白酶激活的传感器就得到了证明。使用腔肠素的海洋荧光素酶（例如NanoLuc和Gaussia荧光素酶）通常会发出蓝光，对于 除了将（拆分的）萤光素酶应用于各种传感应用之外，萤光素酶的特性（例如颜色，亮度和蛋白质稳定性）继续得到改善。Gaur和他的同事重新设计了高斯荧光素酶，一种通常从细胞中分泌出来的非常明亮且稳定的荧光素酶，可以保留在细胞中，同时仍然可以有效折叠。（13）高斯荧光素酶因此成为一种可行的替代荧光素酶，用于细胞内感测。和成像应用，通过将其开发成用于ER应力和caspase激活的传感器得到了证明。使用腔肠素的海洋荧光素酶（例如NanoLuc和Gaussia荧光素酶）通常会发出蓝光，对于 除了将（拆分的）萤光素酶应用于各种传感应用之外，萤光素酶的特性（例如颜色，亮度和蛋白质稳定性）继续得到改善。Gaur和他的同事重新设计了高斯荧光素酶，一种通常从细胞中分泌出来的非常明亮且稳定的荧光素酶，可以保留在细胞中，同时仍然可以有效折叠。（13）高斯荧光素酶因此成为一种可行的替代荧光素酶，用于细胞内感测。和成像应用，通过将其开发成用于ER应力和caspase激活的传感器得到了证明。使用腔肠素的海洋荧光素酶（例如NanoLuc和Gaussia荧光素酶）通常会发出蓝光，对于 Gaur和他的同事重新设计了高斯荧光素酶，一种通常从细胞中分泌出来的非常明亮且稳定的荧光素酶，可以保留在细胞中，同时仍然可以有效折叠。（13）高斯荧光素酶因此成为一种可行的替代荧光素酶，用于细胞内感测。和成像应用，通过将其开发成用于ER应力和caspase激活的传感器得到了证明。使用腔肠素的海洋荧光素酶（例如NanoLuc和Gaussia荧光素酶）通常会发出蓝光，对于 Gaur和他的同事重新设计了高斯荧光素酶，一种通常从细胞中分泌出来的非常明亮且稳定的荧光素酶，可以保留在细胞中，同时仍然可以有效折叠。（13）高斯荧光素酶因此成为一种可行的替代荧光素酶，用于细胞内感测。和成像应用，通过将其开发成用于ER应力和caspase激活的传感器得到了证明。使用腔肠素的海洋荧光素酶（例如NanoLuc和Gaussia荧光素酶）通常会发出蓝光，对于 通过将其开发成用于内质网应激和胱天蛋白酶激活的传感器得到了证明。使用腔肠素的海洋荧光素酶（例如NanoLuc和Gaussia荧光素酶）通常会发出蓝光，对于 通过将其开发成用于内质网应激和胱天蛋白酶激活的传感器得到了证明。使用腔肠素的海洋荧光素酶（例如NanoLuc和Gaussia荧光素酶）通常会发出蓝光，对于体内应用。Ai小组最近报告了一系列带有红移发射的吡啶基官能化底物变体的开发，并使用定向进化产生了具有广泛底物特异性的LumiLuc荧光素酶变体，可以容纳这些底物。（14）尽管蛋白质的重要​​性在生物发光和荧光传感器蛋白质领域中，工程学是显而易见的，蛋白质工程在许多其他传感器格式中也起着关键作用。O'Neill和Lauglin报道了神经元的Ca遗传编码的记录系统的结构²⁺基于所述尖峰的Ca ²⁺-触发分裂的蛋白酶系统的重建。（15）蛋白酶活性通过笼状光学报道分子的裂解来监测，但是该系统的模块化也使其可以与其他下游过程连接。几种类型的生物纳米孔蛋白已成功用于下一代DNA测序和其他传感应用。Long小组的工作表明，选择好的赖氨酸通道蛋白入口或管腔内带电残基的单点突变可以增强赖氨酸生物传感器的灵敏度和选择性。（16） Liu及其同事报道了天然通道蛋白，他的目标是通过将wt蛋白中的22条链增加至34条来进一步增加β-桶状蛋白羟酸铁摄取蛋白组分A（FhuA）的通道直径。（17）孔径的增加是实际上，总共观察到多达30条β链，而插入其他β链则导致孔塌陷并减小了有效通道直径。许多利用生物传感器的研究已经发表在ACS合成生物学。例如，细菌转录因子在复杂的生物回路工程中起着至关重要的作用。Silva-Rocha等人利用与传感器和定点诱变的分子对接研究。设计了新的转录因子，增强了对某些分子（如苯甲酸酯和水杨酸）的反应，以诱导大肠杆菌中的基因表达。（18）在另一项研究中，菲尔德斯及其同事最近报道了酿酒酵母的产生地高辛配基和孕激素生物传感器基于不稳定的二聚体配体结合结构域，该结构域会经历配体诱导的稳定作用。（19）这些生物传感器将为将来构建逻辑门和其他潜在应用提供更大的灵活性。在另一项研究中，Alexandrovov及其同事使用SpyTag / SpyCatcher介导的蛋白连接来设计模块化组织的，依赖支架的蛋白酶传感器，以更好地理解蛋白酶诱导的蛋白与蛋白之间的相互作用。（20）通过这种方式，一套集成的信号传感和扩增功能建立了可以检测α-凝血酶和前列腺特异性抗原活性的电路。生物传感器在光遗传学中也起着关键作用，该领域的研究人员将光与基因工程的感光体结合在一起，以精确地控制某些生物过程。在最近的一篇文章中，Tabor等人。设计了一本小说大肠杆菌近红外光传感器。（21）与文献中的其他系统不同，它们的新系统不需要任何辅助信使，从而简化了过程并实现了快速响应动态。在另一项光遗传学研究中，Weber及其同事设计了一种基于光敏细菌转录因子CarH及其嗜热链球菌同源DNA操纵子序列CarO的系统，以控制哺乳动物细胞中的基因表达。（22）传感器的构建还依赖于生物共轭物的化学结构，并在多篇文章生物共轭化学已经描述了这类工作。生物传感器的重要用途之一是蛋白质稳态的研究，这是细胞应激破坏蛋白质稳态的过程。Fares等。开发了一种荧光蛋白质组应力传感器，该传感器可用于监测蛋白质折叠并提供有关细胞应力如何影响蛋白质组的见识。（23）当与标准免疫球蛋白结合时，Ackerman等人。表明氟激活蛋白可用于体外抗原检测和细胞消融（24）在可用于高通量筛选技术的特定调节剂的开发中存在一些局限性。例如，Sakyiamah及其同事设计了一种新颖的基于NanoBRET的传感器，可用于筛选CXCR4配体。（25）由于CXCR4在许多病原体（如HIV）中发挥作用，因此这种改进的方法可能会导致新的针对CXCR4的药物未来。在另一项研究中，艾哈迈德（Ahmed）等人。他们开发了一种FRET传感器来可视化小RNA的传递和释放。（26）他们的发现表明，小RNA传递载体可能有助于更好地理解非编码RNA生物学。接下来，作为一种新型的萤光素酶，NanoLuc的亮度是传统萤光素酶的100倍，并且由于其体积小，对蛋白质功能的影响很小。在最近的一项研究中，Du等人。设计了一种基因编码的生物传感器，体外，将来可能会翻译成小鼠。本期《虚拟问题》中的文章代表了蛋白质工程和合成生物学如何在传感器领域做出贡献的有说服力的示例。蛋白质工程技术通过改进的功能性实现了生物传感领域的许多突破，我们坚信蛋白质工程技术将继续在新的生物传感器技术的开发中发挥关键作用。本社论中表达的观点只是作者的观点，不一定是ACS的观点。本文引用了其他27个出版物。