稀土离子跨人血红细胞膜的荧光法研究

采用Fura-2荧光浓度指示剂对红细胞的稀土跨膜作用进行了系列研究.结果表明,稀土离子不能通过完整的红细胞膜进入细胞内.通过与离子载体实验相对照,发现细胞ATP耗竭后,低浓度的稀土离子(5×10^-6mol/L)不能跨膜进入ATP-耗竭红细胞.KCl去极化及加入电压依赖性钙通道刺激剂Bay-K8644对稀土离子的跨膜也没有促进作用.在Ca²⁺内流正常的情况下,低浓度稀土离子(5×10^-6mol/L)对钙离子内流无影响.增大稀土离子浓度到5×10^-4mol/L,用显微镜观察此时红细胞已开始溶血.在模拟胞内离子组分的缓冲液中(pH=7.05),比较了La³⁺,Eu³⁺和Ca²⁺对Fura-2的敏感程度.此条件下Fura-2对La³⁺和Eu³⁺的检测限分别为10-12和10-14mol/L,对Ca²⁺的检测限为10-8mol/L,并测得Fura-2-La³⁺(Eu³⁺)的络合比为1∶1,表观离解常数为1.7×10^-12和4.95×10^-14mol/L,表明用此法检测稀土离子跨膜行为相当灵敏有效.     

Recombinant firefly luciferase in Escherichia coli

The authentic recombinant luciferase, the luciferase with the structure similar to that of the native protein, was obtained using random mutagenesis, and its properties were studied in comparison with several fusion proteins. Thermoinactivation curves of the recombinant luciferases within the 10–50°C temperature interval showed that thermoinactivation involves reversible and irreversible steps. Immobilization of the recombinant Luciola mingrelica and Photinus pyralis firefly luciferases on BrCN-activated sepharose was carried out. Immobilization resulted in the preparation of enzymes with high catalytic activity. Physicochemical properties and analytical characteristics of the immobilized recombinant and native luciferases were studied. The catalytic properties of the immobilized recombinant L. mingrelica luciferase were close to those of the native luciferase but the former enzyme appeared to be significantly more stable. The immobilized recombinant luciferases can be used for ATP assay within 0.01–10000 nM range.     

三磷酸腺苷酶的化学模拟

综述了ATP酶的化学模拟的研究进展。     

Die Identifizierung der Natrium-Kalium-Pumpe (Nobel-Vortrag)

Zu provokativ erschien J. C. Skou der Begriff „Pumpe”︁ für den Titel seiner Publikation „The Influence of Some Cations on an Adenosine Triphosphatase from Peripheral Nerves”︁, in der er 1957 beschrieb, daß eine ATPase in der Membran von Krabbennerven das Substrat ATP und den gerichteten Transport von Na- und K-Ionen in charakteristischer Weise beeinflußt. Diese Charakteristika ließen keinen anderen Schluß zu, als daß dieses Enzym, das heute ganz selbstverständlich als „Na⁺-K⁺-Pumpe” bezeichnet wird, am aktiven Transport von Na⁺ durch die Zellmembran beteiligt ist.     

基于纳米金的ATP检测体系的研究进展

ATP是机体代谢过程中不可或缺的能量物质，参与机体的各项重要的生理生化活动，在各方面起着至关重要的作用。因此，对ATP的快速精准检测具有重要的临床意义。纳米金（AuNPs）因优良的光学特性、催化活性、生物相容性等特点，被广泛应用于构建ATP检测体系。本文综述了目前AuNPs应用于ATP检测的研究进展，如比色检测、荧光分析、电化学检测、电化学发光检测、化学发光检测以及表面增强拉曼散射等，比较了各种检测方法的优点与不足，并对AuNPs在ATP检测应用中面临的挑战和机遇做了展望。     

Overcoming Debye length limitations: Three-dimensional wrinkled graphene field-effect transistor for ultra-sensitive adenosine triphosphate detection

Adenosine triphosphate (ATP) is closely related to the pathogenesis of certain diseases, so the detection of trace ATP is of great significance to disease diagnosis and drug development. Graphene field-effect transistors (GFETs) have been proven to be a promising platform for the rapid and accurate detection of small molecules, while the Debye shielding limits the sensitive detection in real samples. Here, a three-dimensional wrinkled graphene field-effect transistor (3D WG-FET) biosensor for ultra-sensitive detection of ATP is demonstrated. The lowest detection limit of 3D WG-FET for analyzing ATP is down to 3.01 aM, which is much lower than the reported results. In addition, the 3D WG-FET biosensor shows a good linear electrical response to ATP concentrations in a broad range of detection from 10 aM to 10 pM. Meanwhile, we achieved ultra-sensitive (LOD: 10 aM) and quantitative (range from 10 aM to 100 fM) measurements of ATP in human serum. The 3D WG-FET also exhibits high specificity. This work may provide a novel approach to improve the sensitivity for the detection of ATP in complex biological matrix, showing a broad application value for early clinical diagnosis and food health monitoring.     

A Small-Molecule Fluorescence Probe for Nuclear ATP

Fluorescence monitoring of ATP in different organelles is now feasible with a few biosensors developed, which, however, show low sensitivity, limited biocompatibility, and accessibility. Small-molecule ATP probes that alleviate those limitations thus have received much attention recently, leading to a few ATP probes that target several organelles except for the nucleus. We disclose the first small-molecule probe that selectively detects nuclear ATP through reversible binding, with 25-fold fluorescence enhancement at pH 7.4 and excellent selectivity against various biologically relevant species. Using the probe, we observed 2.1–3.3-fold and 3.9–7.8-fold higher nuclear ATP levels in cancerous cell lines and tumor tissues compared with normal cell lines and tissues, respectively, which are explained by the higher nuclear ATP level in the mitosis phase. The probe has great potential for studying nuclear ATP-associated biology.     

Light-Harvesting Artificial Cells for the Generation of ATP and NADPH†

The bottom-up construction of self-powered artificial cells is significant to understand the energy supply and metabolism of nature cells. Here, we demonstrate an efficient manner to build thylakoid-containing artificial cells, which continuously convert light energy into chemical energy to supply adenosine 5’-triphosphate (ATP) under light illumination. The production of ATP supplies energy to promote the biological enzyme cascade reactions, where glucose is transformed into glucose 6-phosphate (G6P) under the catalysis of hexokinase (HK). G6P was further converted to gluconolactone 6-phosphate (PG) in the presence of 6-phosphate dehydrogenase (G6PDH), meanwhile NADP⁺ was converted to nicotinamide adenine dinucleotide phosphate (NADPH). The self-powered artificial cells were demonstrated to generate ATP and NADPH successively, which provided a way for building more complicated artificial cells.