Recent Advances in the Development of Optical Imaging Probes for γ-Glutamyltranspeptidase

γ-Glutamyltranspeptidase (GGT) is a cell-membrane-bound protease that participates in cellular glutathione and cysteine homeostasis, which are closely related to many physiological and pathological processes. The accurate measurement of GGT activity is useful for the early diagnosis of diseases. In the past few years, many efforts have been made to build optical imaging probes for the detection of GGT activity both in vitro and in vivo. In this Minireview, recent advances in the development of various optical imaging probes for GGT, including activatable fluorescence probes, ratiometric fluorescence probes, and activatable bioluminescence probes, are summarized. This review starts from the instruction of the GGT enzyme and its biological functions, followed by a discussion of activatable fluorescence probes that show off–on fluorescence in response to GGT. GGT-activatable two-photon fluorescence imaging probes with improved imaging depth and spatial resolution are also discussed. Ratiometric fluorescence probes capable of accurately reporting on GGT levels through a self-calibration mechanism are discussed, followed by describing GGT-activatable bioluminescence probes that can offer a high signal-to-background ratio to detect GGT in living mice. Finally, current challenges and further perspectives for the development of molecular imaging probes for GGT are addressed.     

A New Ultrasensitive Bioluminescence-Based Method for Assaying Monoacylglycerol Lipase

A novel bioluminescent Monoacylglycerol lipase (MAGL) substrate 6-O-arachidonoylluciferin, a D-luciferin derivative, was synthesized, physico-chemically characterized, and used as highly sensitive substrate for MAGL in an assay developed for this purpose. We present here a new method based on the enzymatic cleavage of arachidonic acid with luciferin release using human Monoacylglycerol lipase (hMAGL) followed by its reaction with a chimeric luciferase, PLG2, to produce bioluminescence. Enzymatic cleavage of the new substrate by MAGL was demonstrated, and kinetic constants Km and Vmax were determined. 6-O-arachidonoylluciferin has proved to be a highly sensitive substrate for MAGL. The bioluminescence assay (LOD 90 pM, LOQ 300 pM) is much more sensitive and should suffer fewer biological interferences in cells lysate applications than typical fluorometric methods. The assay was validated for the identification and characterization of MAGL modulators using the well-known MAGL inhibitor JZL184. The use of PLG2 displaying distinct bioluminescence color and kinetics may offer a highly desirable opportunity to extend the range of applications to cell-based assays.     

Brominated Luciferins Are Versatile Bioluminescent Probes

We report a set of brominated luciferins for bioluminescence imaging. These regioisomeric scaffolds were accessed by using a common synthetic route. All analogues produced light with firefly luciferase, although varying levels of emission were observed. Differences in photon output were analyzed by computation and photophysical measurements. The brightest brominated luciferin was further evaluated in cell and animal models. At low doses, the analogue outperformed the native substrate in cells. The remaining luciferins, although weak emitters with firefly luciferase, were inherently capable of light production and thus potential substrates for orthogonal mutant enzymes.     

大肠杆菌特异性快速检测系统研制

结合三磷酸腺苷(ATP)生物发光原理和免疫磁分离技术,研制了一种细菌特异性快速检测系统,可实现对细菌的特异性高灵敏度快速检测.该仪器通过改进光学检测模块的避光设计和放大采集电路的噪声抑制设计,及严格控制试剂反应条件,降低了检测背景,从而达到了更低的检测下限并提高了检测灵敏度.对大肠肝菌(ATCC25922)标准菌溶液进行检测,检测下限达到18 CFU/mL, 与培养计数法检测结果相关系数R达到0.993,选取一份样品进行重复性检测,相对平均偏差(R.A.D.)为6.48 %,变异系数(CV)为9.33 %.结合免疫磁分离技术,对混入的高浓度金黄色葡萄球菌的大肠杆菌溶液进行检测,检测结果与原始大肠肝菌培养计数结果一致,证明该仪器及方法对检测大肠肝菌具有良好的特异性.该系统检测速度快,准确度较高,重复性良好,特异性好,可用于食品及环境中的致病菌快速检测.     

基于微通道的ZnO纳米棒生物荧光检测研究

利用种子法和水热合成技术,分别在常规条件下和阵列式微通道中制备氧化锌(ZnO)纳米棒.采用扫描电子显微镜(SEM)、X射线衍射(XRD)等分析方法表征ZnO纳米棒的表面形貌特点和晶体结构.结果表明:微通道中制备的ZnO纳米棒的比表面积、结晶度和c轴取向性均有较大程度的提高.同时,建立了基于阵列式微通道的ZnO纳米棒生物荧光检测方法,利用ZnO纳米棒可显著增强荧光信号,对异硫氰酸荧光素标记的羊抗牛IgG抗体的检测限为1×10-4 μg/mL.     

Monitoring TRPC7 Conformational Changes by BRET Following GPCR Activation

Transient receptor potential canonical (TRPC) channels are membrane proteins involved in regulating Ca²⁺ homeostasis, and whose functions are modulated by G protein-coupled receptors (GPCR). In this study, we developed bioluminescent resonance energy transfer (BRET) biosensors to better study channel conformational changes following receptor activation. For this study, two intramolecular biosensors, GFP10-TRPC7-RLucII and RLucII-TRPC7-GFP10, were constructed and were assessed following the activation of various GPCRs. We first transiently expressed receptors and the biosensors in HEK293 cells, and BRET levels were measured following agonist stimulation of GPCRs. The activation of GPCRs that engage Gα_q led to a Gα_q-dependent BRET response of the functional TRPC7 biosensor. Focusing on the Angiotensin II type-1 receptor (AT₁R), GFP10-TRPC7-RLucII was tested in rat neonatal cardiac fibroblasts, expressing endogenous AT₁R and TRPC7. We detected similar BRET responses in these cells, thus validating the use of the biosensor in physiological conditions. Taken together, our results suggest that activation of Gα_q-coupled receptors induce conformational changes in a novel and functional TRPC7 BRET biosensor.     

基于生物发光技术的细菌总数快速检测仪

针对细菌总数现场检测需求，设计了一种基于ATP（三磷酸腺苷）生物发光技术的细菌总数现场快速检测仪。传统方法检测细菌总数需要2-3天，而利用ATP生物发光技术可在30min之内完成检测，大大提高了检测速度。本检测仪选取H5773-02型光电倍增管作为光电转换器件以检测微弱光信号，该光电倍增管的工作波长范围为330-850nm，峰值检测波长为500nm，ATP生物发光波长为550nm，两者基本一致，使检测仪具有较高的测量灵敏度。采用流动注射技术，在加入被测样品后分别向样品池中加入检测试剂。由于样品池被密闭在检测腔中，因此可有效减小外界光对测量结果的影响，同时也减小了人为加样对检测重复性的影响，提高检测重复性。利用本检测仪对浓度在10^-6-10^-14mol/L内的ATP样品进行了检测。实验表明，测量结果具有明显梯度，测试时间小于400s时，与标准样品浓度间的线性相关系数达到0.986。设计的细菌总数快速检测仪具有检测速度快、重复性高、操作简单等优点，可适用于食品卫生与安全、环境检测等领域。     

Study on rapid quantitative detection of total bacterial counts by the ATP‐bioluminescence and application in probiotic products

The objective of this study was to develop a rapid quantitative detection method by triphosphate (ATP)‐bioluminescence and determine the feasibility to assay total bacterial counts (TBC) in probiotic products. A useful pretreatment technique to eliminate the interference to the ATP‐bioluminescence method was developed, resulting in a 10–100‐fold improvement in the rapid quantitative detection method by ATP‐bioluminescence. The TBC obtained by the ATP‐bioluminescence rapid detection method was tested against the direct microscopic count method or the plate count method and validated on three ATP Assay Systems. The results generated by the ATP methods and the plate count method were comparable to each other when the interference due to non‐microbial cell ATP and matter such as pigment were removed. This study reports the optimisation of an ATP‐bioluminescence assay using a pretreatment technique to eliminate interference in order to quickly determine the viable counts of bacteria in solid and liquid probiotic products.