Interfacial Stereoelectronic Effect Induced by Anchoring Orientation.

Heterogeneous interfaces in most devices play a key role in the material performance. Exploring the atomic structure and electronic properties of metal-molecule interfaces is critical for various potential applications, such as surface sensing, molecular recognition, and molecular electronic devices. This study unveils a ubiquitous interfacial stereoelectronic effect in conjugated molecular junctions by combining first-principles simulation and scanning tunneling microscopy break junction technology. Single-molecule junctions with same-side interfacial anchoring (cis configuration) exhibit higher conductance than those with opposite-side interfacial anchoring (trans configuration). The cis and trans configurations can undergo reversible conversions, resulting in a conductance switching. The stability of these configurations can be adjusted by an electric field, achieving precise regulation of conductance states. Our findings provide important insights for designing high-quality materials and enhancing the device performance.

Exceptional Field Effect and Negative Differential Conductance in Spiro-Conjugated Single-Molecule Junctions.

The advancement of molecular electronics endeavors to build miniaturized electronic devices using molecules as the key building blocks by harnessing their internal structures and electronic orbitals. To date, linear planar conjugated or cross-conjugated molecules have been extensively employed in the fabrication of single-molecule devices, benefiting from their good conductivity and compatibility with electrode architectures. However, the development of multifunctional single-molecule devices, particularly those with unique charge transport properties, necessitates a more rigorous selection of molecular materials. Among different assortments of molecules suited for the construction of molecular circuits, Spiro-conjugated structures, specifically spirobifluorene derivatives, stand out as promising candidates due to their distinctive electronic properties. In this work, we focus on the charge transport characteristics of Spiro-conjugated molecules sandwiched between graphene nanogaps. Experiments reveal significant Coulomb blockade and distinct negative differential conductance effects. Beyond two-terminal device measurements, solid-state gate electrodes are utilized to create single-molecule transistors, successfully modulating the molecular energy levels to achieve an on/off ratio exceeding 1000. This endeavor not only offers valuable insights into the design and fabrication of future practical molecular devices, blessed with enhanced performance and functionality, but also presents a new paradigm for the investigation of fundamental physical phenomena.

In Situ Detection of Interfacial Ions at the Single-Bond Level.

Detecting the ionic state at the solid-liquid interface is essential to reveal the various chemical and physical processes that occur at the interface. In this study, the adsorption states of the highly electronegative ions F- and OH- at the solid-liquid interface are detected by using the scanning tunneling microscopy break junction technique. With the active hydrogen atom of the amino group as a probe, the formed ionic hydrogen bonds are successfully detected, thereby enabling in situ monitoring of the ionic state at the solid-liquid interface. Through noise power spectral density analysis and theoretical simulations, we reveal the mechanism by which ionic hydrogen bonds at the interface affect the charge transport properties. In addition, we discover that the ionic state at the solid-liquid interface can be effectively manipulated by electric fields. Under high electric fields, the concentration of the anion near the electrode is higher, and the proportion of hydrogen bonds formed is greater than that under low electric fields. This study of the interfacial ionic state at the single-bond level provides guidance for the design of high-performance materials for energy conversion and environmental purification.

Real-Time Direct Monitoring of Chirality Fixation and Recognition at the Single-Molecule Level.

Chirality, a fundamental attribute of nature, significantly influences a wide range of phenomena related to physical properties, chemical reactions, biological pharmacology, and so on. As a pivotal aspect of chirality research, chirality recognition contributes to the synthesis of complex chiral products from simple chiral compounds and exhibits intricate interplay between chiral materials. However, macroscopic detection technologies cannot unveil the dynamic process and intrinsic mechanisms of single-molecule chirality recognition. Herein, we present a single-molecule detection platform based on graphene-molecule-graphene single-molecule junctions to measure the chirality recognition involving interactions between amines and chiral alcohols. This approach leads to the realization of in situ and real-time direct observation of chirality recognition at the single-molecule level, demonstrating that chiral alcohols exhibit compelling potential to induce the formation of the corresponding chiral configuration of molecules. The amalgamation of theoretical analyses with experimental findings reveals a synergistic action between electrostatic interactions and steric hindrance effects in the chirality recognition process, thus substantiating the microscopic mechanism governing the chiral structure-activity relationship. These studies open up a pathway for exploring novel chiral phenomena from the fundamental limits of chemistry, such as chiral origin and chiral amplification, and offer important insights into the precise synthesis of chiral materials.

Tracking Noncovalent Interactions of π, π-Hole, and Ion in Molecular Complexes at the Single-Molecule Level.

Noncovalent interactions involving aromatic rings, such as π-stacking and π-ion interactions, play an essential role in molecular recognition, assembly, catalysis, and electronics. However, the inherently weak and complex nature of these interactions has made it challenging to study them experimentally, especially with regard to elucidating their properties in solution. Herein, the noncovalent interactions between π and π-hole, π and cation, and π-hole and anion in molecular complexes in nonpolar solution are investigated in situ through single-molecule electrical measurements in combination with theoretical calculations. Specifically, phenyl and pentafluorobenzyl groups serve as π and π-hole sites, respectively, while Li+ and Cl- are employed as the cation and anion. Our findings reveal that, in comparison with homogeneous π···π interactions, heterogeneous π···π-hole and π···cation interactions exhibit greater binding energies, resulting in a longer binding lifetime of the molecular junctions. Meanwhile, π···Li+ and π-hole···Cl- interactions present significantly distinct binding characteristics, with the former being stronger but more flexible than the latter. Furthermore, by changing the molecular components, similar conductivity can be achieved in both molecular dimers or sandwich complexes. These results provide new insights into π- and π-hole-involved noncovalent interactions, offering novel strategies for precise manipulation of molecular assembly, recognition, and molecular device.

Electronic Devices Based on Heterostructures of 2D Materials and Self-Assembled Monolayers.

2D materials (2DMs), known for their atomically ultrathin structure, exhibit remarkable electrical and optical properties. Similarly, molecular self-assembled monolayers (SAMs) with comparable atomic thickness show an abundance of designable structures and properties. The strategy of constructing electronic devices through unique heterostructures formed by van der Waals assembly between 2DMs and molecular SAMs not only enables device miniaturization, but also allows for convenient adjustment of their structures and functions. In this review, the fundamental structures and fabrication methods of three different types of electronic devices dominated by 2DM-SAM heterojunctions with varying architectures are timely elaborated. Based on these heterojunctions, their fundamental functionalities and characteristics, as well as the regulation of their performance by external stimuli, are further discussed.

Methylation-regulated tumor suppressor gene PDE7B promotes HCC invasion and metastasis through the PI3K/AKT signaling pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) has a high mortality rate, and the mechanisms underlying tumor development and progression remain unclear. However, inactivated tumor suppressor genes might play key roles. DNA methylation is a critical regulatory mechanism for inactivating tumor suppressor genes in HCC. Therefore, this study investigated methylation-related tumor suppressors in HCC to identify potential biomarkers and therapeutic targets. METHODS: We assessed genome-wide DNA methylation in HCC using whole genome bisulfite sequencing (WGBS) and RNA sequencing, respectively, and identified the differential expression of methylation-related genes, and finally screened phosphodiesterase 7B (PDE7B) for the study. The correlation between PDE7B expression and clinical features was then assessed. We then analyzed the changes of PDE7B expression in HCC cells before and after DNA methyltransferase inhibitor treatment by MassArray nucleic acid mass spectrometry. Furthermore, HCC cell lines overexpressing PDE7B were constructed to investigate its effect on HCC cell function. Finally, GO and KEGG were applied for the enrichment analysis of PDE7B-related pathways, and their effects on the expression of pathway proteins and EMT-related factors in HCC cells were preliminarily explored. RESULTS: HCC exhibited a genome-wide hypomethylation pattern. We screened 713 hypomethylated and 362 hypermethylated mCG regions in HCC and adjacent normal tissues. GO analysis showed that the main molecular functions of hypermethylation and hypomethylation were "DNA-binding transcriptional activator activity" and "structural component of ribosomes", respectively, whereas KEGG analysis showed that they were enriched in "bile secretion" and "Ras-associated protein-1 (Rap1) signaling pathway", respectively. PDE7B expression was significantly down-regulated in HCC tissues, and this low expression was negatively correlated with recurrence and prognosis of HCC. In addition, DNA methylation regulates PDE7B expression in HCC. On the contrary, overexpression of PDE7B inhibited tumor proliferation and metastasis in vitro. In addition, PDE7B-related genes were mainly enriched in the PI3K/ATK signaling pathway, and PDE7B overexpression inhibited the progression of PI3K/ATK signaling pathway-related proteins and EMT. CONCLUSION: PDE7B expression in HCC may be regulated by promoter methylation. PDE7B can regulate the EMT process in HCC cells through the PI3K/AKT pathway, which in turn affects HCC metastasis and invasion.

Unveiling the Properties of Sulfhydryl Groups in a Single-Molecule Junction.

The metal-thiol interface is ubiquitous in nanotechnology and surface chemistry. It is not only used to construct nanocomposites but also plays a decisive role in the properties of these materials. When organothiol molecules bind to the gold surface, there is still controversy over whether sulfhydryl groups can form disulfide bonds and whether these disulfide bonds can remain stable on the gold surface. Here, we investigate the intrinsic properties of sulfhydryl groups on the gold surface at the single-molecule level using a scanning tunneling microscope break junction technique. Our findings indicate that sulfhydryl groups can react with each other to form disulfide bonds on the gold surface, and the electric field can promote the sulfhydryl coupling reaction. In addition to these findings, ultraviolet irradiation is used to effectively regulate the coupling between sulfhydryl groups, leading to the formation and cleavage of disulfide bonds. These results unveil the intrinsic properties of sulfhydryl groups on the gold surface, therefore facilitating the accurate construction of broad nanocomposites with the desired functionalities.

Recent Advances in Flexible Wearable Technology: From Textile Fibers to Devices.

Smart textile fabrics have been widely investigated and used in flexible wearable electronics because of their unique structure, flexibility and breathability, which are highly desirable with integrated multifunctionality. Recent years have witnessed the rapid development of textile fiber-based flexible wearable devices. However, the pristine textile fibers still can't meet the high standards for practical flexible wearable devices, which calls for the development of some effective modification strategies. In this review, we summarize the recent advances in the flexible wearable devices based on the textile fibers, putting special emphasis on the design and modifications of textile fibers. In addition, the applications of textile fibers in various fields and the critical role of textile fibers are also systematically discussed, which include the supercapacitors, sensors, triboelectric nanogenerators, thermoelectrics, and other self-powered electronic devices. Finally, the main challenges that should be overcome and some effective solutions are also manifested, which will guide the future development of more effective textile fiber-based flexible wearable devices.

Mechanistic Insight into Lewis Acid-Catalyzed Cycloaddition of Bicyclo[1.1.0]butanes with Ketene: Bicyclo[1.1.0]butanes Serving as an Electrophile.

Lewis acid-catalyzed cycloaddition between bicyclo[1.1.0]butanes (BCBs) and unsaturated substrates has recently been demonstrated to be a powerful strategy for synthesizing bicyclo[2.1.1]hexanes. However, their reaction mechanisms remain elusive. This computational work explored the recently developed TMSOTf-catalyzed cycloaddition of BCB ketone to ketene and determined the rate-determining step as the activation of BCB ketone. Contrary to the previous proposal of BCB enolate as the active species, this work instead identified the catalytically active species to be a partially Lewis acid-activated BCB cation, which shows a greater electrophilicity and larger orbital interactions with ketene compared to those of the pristine BCB. The most favorable reaction pathway uniquely utilizes this activated BCB species as an electrophile to react with ketene as a nucleophile, while the previously proposed enolate is relatively inactive. Moreover, the in situ-generated TfO anion is revealed to be non-innocent, and its coordination mode and orientation could affect the reaction kinetics.

Building Molecular Nanomagnets by Encapsulating Lanthanide Ions in Boron Nitride Nanotubes: Ab Initio Investigation.

Lanthanide-based single-ion magnets have attracted much interest due to their great potential for information storage at the level of one molecule. Among various strategies to enhance magnetization blocking in such complexes, the synthesis of axially symmetric compounds is regarded as the most promising. Here, we investigate theoretically the magnetization blocking of several lanthanide ions (Tb3+, Dy3+, Ho3+, Er3+, and Tm3+) encapsulated in highly symmetric zigzag boron nitride nanotubes (BNNTs) of different diameters with ab initio methodology. We found that Tb3+@(7,0)BNNT, Dy3+@(7,0)BNNT, and Tm3+@(5,0)BNNT are suitable SIM candidates, while the other investigated complexes from this series show no signs of magnetization blocking owing to a hard competition between contributions to the crystal field of the lanthanide ion from neighboring and more distant atoms of the nanotube.

Direct visualization endoscopic retrograde appendicitis therapy for treatment of acute uncomplicated appendicitis.

OBJECTIVE: To investigate the diagnostic and therapeutic value of direct visualization (single-use eyeMax subscope) endoscopic management of acute uncomplicated appendicitis. METHODS: Thirty-six patients diagnosed with acute uncomplicated appendicitis, confirmed by computed tomography or ultrasonography, from Jan 2023 to Feb 2024 were enrolled in this study. We collected demographics, colonoscopy findings, subscope findings, clinical outcomes of endoscopic retrograde appendicitis therapy (ERAT), and adverse events associated with ERAT. RESULTS: Appendiceal intubation was successful for all 36 patients. Thirty-five patients (97.2%) were definitely confirmed as having acute uncomplicated appendicitis. One patient with negative appendicitis was diagnosed as having cecal diverticulitis with fecalith incarceration. The mean procedure time was 13.1 ± 13.6 min. One patient presented with worsening abdominal pain, and a computed tomography scan suggested a perforated appendix. The mean length of hospitalizations was 1.78 ± 2 days. The mean follow-up was 158 days; during this period, two patients (5.6%) experienced recurrent abdominal pain after 23 and 88 days and subsequently underwent laparoscopic appendectomy. CONCLUSION: Direct visualization ERAT may be effective for diagnosing and treating acute uncomplicated appendicitis and seems to have a low complication rate.

A Novel lncRNA lncRNA-4045 Promotes the Progression of Hepatocellular Carcinoma by Affecting the Expression of AKR1B10.

BACKGROUND: Long noncoding RNAs (lncRNAs) have been shown to be related to the occurrence and development of a variety of cancers including hepatocellular carcinoma (HCC). However, a large number of potential HCC-related lncRNAs remain undiscovered and are yet to be fully understood. METHODS: Differentially expressed lncRNAs were first obtained from the tumor tissues and adjacent normal tissues of five HCC patients using high-throughput microarray chips. Then the expression levels of 10 differentially expressed lncRNAs were verified in 50 pairs of tissue samples from patients with HCC by quantitative real-time PCR (qRT-PCR). The oncogenic effects of lncRNA-4045 (ENST00000524045.6) in HCC cell lines were verified through a series of in vitro experiments including CCK-8 assay, plate clone formation assay, transwell assay, scratch assay, and flow cytometry. Subsequently, the potential target genes of lncRNA-4045 were predicted by bioinformatics analysis, fluorescence in situ hybridization assay, and RNA sequencing. The mechanism of lncRNA-4045 in HCC was explored by WB assay as well as rescue and enhancement experiments. RESULTS: The results from microarray chips showed 1,708 lncRNAs to have been significantly upregulated and 2725 lncRNAs to have been significantly downregulated in HCC tissues. Via validation in 50 HCC patients, a novel lncRNA lncRNA-4045 was found significantly upregulated in HCC tissues. Additionally, a series of in vitro experiments showed that lncRNA-4045 promoted the proliferation, invasion, and migration of HCC cell lines, and inhibited the apoptosis of HCC cell lines. The results of qRT-PCR in HCC tissues showed that the expression levels of AKR1B10 were significantly positively correlated with lncRNA-4045. LncRNA-4045 knockdown significantly down-regulated AKR1B10 protein expression, and overexpression of lncRNA-4045 led to significant up-regulation of AKR1B10 protein in HCC cell lines. Lastly, down-regulation of AKR1B10 could partially eliminate the enhancement of cell proliferation induced by lncRNA-4045 overexpression, while up-regulation of AKR1B10 was shown to enhance those effects. CONCLUSION: LncRNA-4045 may promote HCC via enhancement of the expression of AKR1B10 protein.

GhGASA14 regulates the flowering time of upland cotton in response to GA3.

KEY MESSAGE: The silencing of GhGASA14 and the identification of superior allelic variation in its coding region indicate that GhGASA14 may positively regulate flowering and the response to GA3. Gibberellic acid-stimulated Arabidopsis (GASA), a member of the gibberellin-regulated short amino acid family, has been extensively investigated in several plant species and found to be critical for plant growth and development. However, research on this topic in cotton has been limited. In this study, we identified 38 GhGASAs that were dispersed across 18 chromosomes in upland cotton, and all of these genes had a GASA core domain. Transcriptome expression patterns and qRT-PCR results revealed that GhGASA9 and GhGASA14 exhibited upregulated expression not only in the floral organs but also in the leaves of early-maturing cultivars. The two genes were functionally characterized by virus-induced gene silencing (VIGS), and the budding and flowering times after silencing the target genes were later than those of the control (TRV:00). Compared with that in the water-treated group (MOCK), the flowering period of the different fruiting branches in the GA3-treated group was more concentrated. Interestingly, allelic variation was detected in the coding sequence of GhGASA14 between early-maturing and late-maturing accessions, and the frequency of this favorable allele was greater in high-latitude cotton cultivars than in low-latitude ones. Additionally, a significant linear relationship was observed between the expression level of GhGASA14 and flowering time among the 12 upland cotton accessions. Taken together, these results indicated that GhGASA14 may positively regulate flowering time and respond to GA3. These findings could lead to the use of valuable genetic resources for breeding early-maturing cotton cultivars in the future.

Characterization and identification of major flavonoids of bamboo leaf extract by HPLC/ESI-QTOF-MS/MS.

Bamboo leaf extract (BLE) is a pale brown powder extracted from bamboo leaves, and it is listed in the Chinese Standard GB-2760 as a legal and safe food additive. The present study aims to identify and characterize the major flavonoids in BLE. The identification of major flavonoids was carried out using ultra performance liquid chromatography combined with electrospray ionization quadruple time-of-flight tandem mass spectrometry (HPLC/ESI-QTOF-MS/MS). A total of 31 flavonoid compounds were identified and tentatively characterized base on reference standards and MS dissociation mechanisms. HPLC/ESI-QTOF-MS can serve as an important analytical platform to identification structure of bamboo leaf flavonoids (BLF).

Tunable Interferometric Effects between Single-Molecule Suzuki-Miyaura Cross-Couplings.

Parallel two molecular bridges loaded with a palladium catalyst were integrated into separate pairs of graphene electrodes in the same device. Based on the complete description of the one-palladium catalytic pathway by single-molecule electrical spectroscopy, this setup enables the mapping of the cross-correlation between different catalysts and demonstrates the emergent complexity in the extrapolation from single molecule to ensemble. The anticorrelation behaviors at the time scale of two individual catalysts in sufficiently close proximity were revealed in the Suzuki-Miyaura cross-coupling. Further experimental evidence demonstrates that the long-range electric dipole-dipole interaction induced by solvent leads to the destructive interferometric effect of two catalysts. In contrast, the cooperative coupling of the elementary step between two catalysts affords a local acceleration. This new form of reaction dynamics measurement via focusing on multiple molecules with single-event resolution holds great promise to build a bridge between single molecule and ensemble.

Effects of Dietary Nonfibrous Carbohydrate/Neutral Detergent Fiber Ratio on Methanogenic Archaea and Cellulose-Degrading Bacteria in the Rumen of Karakul Sheep: a 16S rRNA Gene Sequencing Study.

The study was conducted to investigate the effects of dietary nonfibrous carbohydrate (NFC)/neutral detergent fiber (NDF) ratio on methanogenic archaea and cellulose-degrading bacteria in Karakul sheep by 16S rRNA gene sequencing. Twelve Karakul sheep were randomly divided into four groups, each group with three replicates, and they were fed with four dietary NFC/NDF ratios at 0.54, 0.96, 1.37, and 1.90 as groups 1, 2, 3, and 4, respectively. The experiment lasted for four periods: I (1 to 18 days), II (19 to 36 days), III (37 to 54 days), and IV (55 to 72 days); during each period, rumen contents were collected before morning feeding to investigate on methanogenic archaea and cellulose-degrading bacteria. The results showed that with an increase in dietary NFC/NDF ratio, the number of rumen archaea operational taxonomic units and the diversity of archaea decrease. The most dominant methanogens did not change with dietary NFC/NDF ratio and prolongation of experimental periods. Methanobrevibacter was the most dominant genus. At the species level, the relative abundance of Methanobrevibacter ruminantium first increased and then decreased when the NFC/NDF ratio increased. When the dietary NFC/NDF ratio was 0.96, the structure of archaea was largely changed, and the relative abundance of Fibrobacter sp. strain UWCM, Ruminococcus flavefaciens, and Ruminococcus albus were the highest. When the dietary NFC/NDF ratio was 1.37, the relative abundance of Butyrivibrio fibrisolvens was higher than for other groups. Based on all the data, we concluded that a dietary NFC/NDF ratio of ca. 0.96 to 1.37 was a suitable ratio to support optimal sheep production. IMPORTANCE CH4 produced by ruminants aggravates the greenhouse effect and cause wastage of feed energy, and CH4 emissions are related to methanogens. According to the current literature, there is a symbiotic relationship between methanogens and cellulolytic bacteria, so reducing methane will inevitably affect the degradation of fiber materials. This experiment used 16S rRNA gene high-throughput sequencing technology to explore the balance relationship between methanogens and cellulolytic bacteria for the first time through a long-term feeding period. The findings provide fundamental data, supporting for the diet structures with potential to reduce CH4 emission.

Diagnostic Accuracy of Left Atrial/Left Atrial Appendage Thrombus in Patients with Atrial Fibrillation: A Systematic Review and Network Meta-Analysis.

Background: This paper aimed to appraise the diagnostic precision of assorted methodologies to identify left atrial/left atrial appendage (LA/LAA) thrombus through a network meta-assessment. Methods: Methodologically, we conducted a comprehensive literature search across multiple databases. Utilizing the risk of bias tool from the Cochrane Collaboration, methodological quality of included studies was critically assessed and potential publication bias was examined via funnel plots. The subsequent data analysis was executed using Stata software, with the most efficacious diagnostic modalities being determined based on cumulative ranking curve (SUCRA) values. Results: We scrutinized a sum of 18 papers, comprising 4102 subjects and utilizing 10 different diagnostic techniques. The hierarchical results derived from the network meta-analysis indicated that in regards to sensitivity, the dual-source cardiac computed tomography (DSCT) was superior (with a SUCRA value of 71.7%), it was succeeded by 3-minute delayed cardiac computed tomography (CCT) (scoring 66.8%), which surpassed the transesophageal echocardiography (TEE) (holding a SUCRA value of 57.5%). In terms of specificity, DSCT was the best (SUCRA value of 84.3%), followed by three dimensional (3D) cardiac magnetic resonance imaging (3D-CMRI) (SUCRA value of 78.0%), which was better than TEE (SUCRA value of 66.6%). In terms of positive likelihood ratio (PLR), 6-minute delayed CCT (SUCRA value of 85.6%) was superior to 3-minute delayed CCT (SUCRA value of 80.1%), both of which were superior to TEE (SUCRA value of 69.1%). DSCT (SUCRA value of 89.3%) had the best negative likelihood ratio (NLR), while DSCT (SUCRA value of 79.9%) had the highest accuracy. Conclusions: This study demonstrated that DSCT outperformed TEE in sensitivity, specificity, NLR, and accuracy in identifying thrombus of LA/LAA among patients suffering from atrial fibrillation. Our conclusion is that DSCT is the best in diagnosing LA/LAA. In addition, 3D-CMRI and 3-minute delayed CCT are expected to replace TEE.

Dipole-Modulated Charge Transport through PNP-Type Single-Molecule Junctions.

The PNP structure realized by energy band engineering is widely used in various electronic and optoelectronic devices. In this work, we succeed in constructing a PNP-type single-molecule junction and explore the intrinsic characteristics of the PNP structure at the single-molecule level. A back-to-back azulene molecule is designed with opposite ∼1.7 D dipole moments to create PNP-type single-molecule junctions. In combination with theoretical and experimental studies, it is found that the intrinsic dipole can effectively adjust single-molecule charge transport and the corresponding potential barriers. This energy band control and charge transport regulation at the single-molecule level improve deep understanding of molecular charge transport mechanisms and provide important insights into the development of high-performance functional molecular nanocircuits toward practical applications.

Accurate Single-Molecule Kinetic Isotope Effects.

An accurate single-molecule kinetic isotope effect (sm-KIE) was applied to circumvent the inherent limitation of conventional ensemble KIE by using graphene-molecule-graphene single-molecule junctions. In situ monitoring of the single-molecule reaction trajectories in real time with high temporal resolution has the capability to characterize the deeper information brought by KIE. The C-O bond cleavage and the C-C bond formation of the transition state (TS) were observed in the Claisen rearrangement through the secondary kinetic isotope effect, demonstrating the high detection sensitivity and accuracy of this method. More interestingly, this sm-KIE can be used to determine TS structures under different electric fields, revealing the multidimensional regulation of the TS. The detection and manipulation of the TS provide a broad perspective to understand and optimize chemical reactions and biomimetic progress.