Digital bioassays based on single-molecule enzyme reactions represent a new class of bioanalytical methods that enable the highly sensitive detection of biomolecules in a quantitative manner. Since ...the first reports of these methods in the 2000s, there has been significant growth in this new bioanalytical strategy. The principal strategy of this method is to compartmentalize target molecules in micron-sized reactors at the single-molecule level and count the number of microreactors showing positive signals originating from the target molecule. A representative application of digital bioassay is the digital enzyme-linked immunosorbent assay (ELISA). Owing to their versatility, various types of digital ELISAs have been actively developed. In addition, some disease markers and viruses possess catalytic activity, and digital bioassays for such enzymes and viruses have, thus, been developed. Currently, with the emergence of new microreactor technologies, the targets of this methodology are expanding from simple enzymes to more complex systems, such as membrane transporters and cell-free gene expression. In addition, multiplex or multiparametric digital bioassays have been developed to assess precisely the heterogeneities in sample molecules/systems that are obscured by ensemble measurements. In this review, we first introduce the basic concepts of digital bioassays and introduce a range of digital bioassays. Finally, we discuss the perspectives of new classes of digital bioassays and emerging fields based on digital bioassay technology.
This tutorial review focuses on the design principles of digital bioassays based on single-molecule enzyme reactions and further introduces their expanded applications from the perspective of next-generation digital bioassays.
(N‐Phenylfluorenylidene)acridane (Ph‐FA) compounds with electron‐withdrawing and ‐donating substituents (H, MeO, Ph, NO2, Br, F) at the para position of the phenyl group were successfully synthesized ...by Barton–Kellogg reactions of N‐aryl thioacridones and diazofluorene. By using the substituent on the nitrogen atom to alter the electronic properties, both the folded and twisted conformers of p‐NO2‐C6H4‐FA could be crystallographically characterized, which enabled the charge transfer from the electron‐donating acridane moiety to the electron‐accepting fluorenylidene moiety to be understood. Ground‐state mechanochromism, thermochromism, vapochromism, and proton‐induced chromism were demonstrated between the folded and twisted conformations of the conformers. Protonation and chemical oxidation of Ph‐FA gave two stable acridinium compounds, namely, the fluorenylacridinium and acridinium radical cations. The present study will contribute to the development of functional dyes and organic semiconductors.
Color control: (N‐Phenylfluorenylidene)acridane compounds with electron‐withdrawing and ‐donating substituents at the para position of the phenyl group were synthesized and the X‐ray crystal structures of both the folded and twisted conformers of the nitro derivative determined. Not only do the compounds show various chromic properties, carrier transport properties are also evident.
Vegetables are rich sources of nutrients such as fiber, minerals, vitamins, and antioxidants. Vegetables also contain various free-form amino acids, which improves their nutritional and palatable ...value. Cooking alters the content of free amino acids in vegetables, which affects their nutritional values. In this study, free amino acid levels were evaluated after cooking vegetables by different methods, boiling, roasting in an oven, and using a microwave. Results showed that many vegetables analyzed contain aspartate and glutamine abundantly. On the other hand, hydroxyproline, cysteine, ornithine and citrulline are the free amino acids existing at low or undetectable levels in all vegetables tested. The total free amino acid content in vegetables tended to decrease after boiling, and almost the same amount of free amino acids was obtained in the cooking liquid. Roasting of vegetables in an oven resulted in an increase in the content of specific amino acids, including γ-aminobutyric acid (GABA). Thus, it is important to choose the right cooking methods to prevent the loss of free amino acids. The results of the present study emphasize the changes in the contents of free amino acids during cooking with methods that are typically used on a daily basis. Our study on the dynamics of free amino acids caused by various cooking methods provides ample information for future nutritional studies.
Abstract
F
1
F
o
ATP synthase interchanges phosphate transfer energy and proton motive force via a rotary catalysis mechanism. Isolated F
1
-ATPase catalytic cores can hydrolyze ATP, passing through ...six intermediate conformational states to generate rotation of their central γ-subunit. Although previous structural studies have contributed greatly to understanding rotary catalysis in the F
1
-ATPase, the structure of an important conformational state (the binding-dwell) has remained elusive. Here, we exploit temperature and time-resolved cryo-electron microscopy to determine the structure of the binding- and catalytic-dwell states of
Bacillus
PS3 F
1
-ATPase. Each state shows three catalytic β-subunits in different conformations, establishing the complete set of six states taken up during the catalytic cycle and providing molecular details for both the ATP binding and hydrolysis strokes. We also identify a potential phosphate-release tunnel that indicates how ADP and phosphate binding are coordinated during synthesis. Overall these findings provide a structural basis for the entire F
1
-ATPase catalytic cycle.
Herein, we report use of Li+@C60TFSI− as a dopant for spiro‐MeOTAD in lead halide perovskite solar cells. This approach gave an air stability nearly 10‐fold that of conventional devices using ...Li+TFSI−. Such high stability is attributed to the hydrophobic nature of Li+@C60TFSI− repelling moisture and absorbing intruding oxygen, thereby protecting the perovskite device from degradation. Furthermore, Li+@C60TFSI− could oxidize spiro‐MeOTAD without the need for oxygen. The encapsulated devices exhibited outstanding air stability for more than 1000 h while illuminated under ambient conditions.
Air stability remains the most challenging limitation to commercialization of perovskite solar cells. Use of conventional Li+TFSI− dopants leads to instability because of its hygroscopic nature. Using a fullerene‐encapsulated Li+@C60 TFSI− dopant gives a 10‐fold enhancement of the air‐stability of perovskite solar cells, through the dopant's hydrophobic nature, instant oxidation of spiro‐MeOTAD, and anti‐oxidation activity.
The F
1
-ATPase is a rotary motor fueled by ATP hydrolysis. Its rotational dynamics have been well characterized using single-molecule rotation assays. While F
1
-ATPases from various species have ...been studied using rotation assays, the standard model for single-molecule studies has been the F
1
-ATPase from thermophilic
Bacillus
sp. PS3, named TF
1
. Single-molecule studies of TF
1
have revealed fundamental features of the F
1
-ATPase, such as the principal stoichiometry of chemo-mechanical coupling (hydrolysis of 3 ATP per turn), torque (approximately 40 pN·nm), and work per hydrolysis reaction (80 pN·nm = 48 kJ/mol), which is nearly equivalent to the free energy of ATP hydrolysis. Rotation assays have also revealed that TF
1
exhibits two stable conformational states during turn: a binding dwell state and a catalytic dwell state. Although many structures of F
1
have been reported, most of them represent the catalytic dwell state or its related states, and the structure of the binding dwell state remained unknown. A recent cryo-EM study on TF
1
revealed the structure of the binding dwell state, providing insights into how F
1
generates torque coupled to ATP hydrolysis. In this review, we discuss the torque generation mechanism of F
1
based on the structure of the binding dwell state and single-molecule studies.
Abstract
ATPase inhibitory factor 1 (IF1) is a mitochondrial regulatory protein that blocks ATP hydrolysis of F1-ATPase, by inserting its N-terminus into the rotor–stator interface of F1-ATPase. ...Although previous studies have proposed a two-step model for IF1-mediated inhibition, the underlying molecular mechanism remains unclear. Here, we analysed the kinetics of IF1-mediated inhibition under a wide range of ATPs and IF1s, using bovine mitochondrial IF1 and F1-ATPase. Typical hyperbolic curves of inhibition rates with IF1s were observed at all ATPs tested, suggesting a two-step mechanism: the initial association of IF1 to F1-ATPase and the locking process, where IF1 blocks rotation by inserting its N-terminus. The initial association was dependent on ATP. Considering two principal rotation dwells, binding dwell and catalytic dwell, in F1-ATPase, this result means that IF1 associates with F1-ATPase in the catalytic-waiting state. In contrast, the isomerization process to the locking state was almost independent of ATP, suggesting that it is also independent of the F1-ATPase state. Further, we investigated the role of Glu30 or Tyr33 of IF1 in the two-step mechanism. Kinetic analysis showed that Glu30 is involved in the isomerization, whereas Tyr33 contributes to the initial association. Based on these findings, we propose an IF1-mediated inhibition scheme.
Graphical Abstract
Which way to go: The product distribution in the efficient oxidation of 2‐aminoanthracene derivatives to pyrazine‐ and pyrrole‐fused bisanthracenes can be controlled by additives (see scheme; ...TFA=trifluoroacetic acid, DDQ=2,3‐dichloro‐5,6‐dicyano‐1,4‐benzoquinone). The pyrrole‐fused dimer can be regarded as an aza7helicene with a stable helical conformation.
Alkaline phosphatase (ALP), a homo‐dimeric enzyme has been widely used in various bioassays as disease markers and enzyme probes. Recent advancements of digital bioassay revolutionized ALP‐based ...diagnostic assays as seen in rapid growth of digital ELISA and the emerging multiplex profiling of single‐molecule ALP isomers. However, the intrinsic heterogeneity found among ALP molecules hampers the ALP‐based quantitative digital bioassays. This study aims quantitative analysis of single‐molecule activities of ALP from Escherichia coli and reveals the static heterogeneity in catalytic activity of ALP with two distinct populations: half‐active and fully‐active portions. Digital assays with serial buffer exchange uncovered single‐molecule Michaelis–Menten kinetics of ALP; half‐active molecules have halved values of the catalytic turnover rate, kcat, and the rate constant of productive binding, kon, of the fully active molecules. These findings suggest that half‐active ALP molecules are heterogenic dimers composed of inactive and active monomer units, while fully active ALP molecules comprise two active units. Static heterogeneity was also observed for ALP with other origins: calf intestine or shrimp, showing how the findings can be generalized across species. Cell‐free expression of ALP with disulfide bond enhancer and spiked zinc ion resulted in homogenous population of ALP of full activity, implying that inactive monomer units of ALP are deficient in correct disulfide bond formation and zinc ion coordination. These findings provide basis for further study on molecular mechanism and biogenesis of ALP, and also offer the way to prepare homogenous and active populations of ALP for highly quantitative and sensitive bioassays with ALP.
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