This study presents a novel loop-mediated isothermal amplification (LAMP) method for the rapid and sensitive detection of porcine DNA, addressing critical needs in food safety and compliance with halal and kosher dietary laws. The key innovation lies in the design of highly specific primers targeting Sus scrofa mitochondrial DNA, ensuring accurate identification with minimal cross-reactivity. The LAMP technique was selected for its rapid amplification, isothermal operation, and simplified equipment requirements, making it suitable for on-site applications and reducing overall costs. Optimization of reaction conditions enabled reliable DNA amplification at 63°C within 60 minutes. The progress of the amplification was monitored in real time by measuring turbidity, while successful DNA synthesis was further confirmed by gel electrophoresis. For sensitive detection and quantification, an electrochemical DNA biosensor was integrated into the workflow. This biosensor utilizes a silicon nanowire–platinum nanoparticle–modified screen-printed carbon electrode and ferrocenylnaphthalene diimide as a dsDNA intercalator, enabling precise, real-time monitoring of the LAMP reaction. The optimized assay achieved a detection limit of 175.2 ng/μL, demonstrating high sensitivity for low-level porcine DNA detection. Specificity was validated across various meat sources and processed foods, with no false positives observed. The biosensor-based method also successfully detected porcine DNA in mixed meat samples, highlighting its strong potential for practical use in food authenticity testing and regulatory compliance.

G-quadruplex (G4) DNA structures are widely distributed in biologically important genomic regions and are involved in gene regulation and genome stability. However, experimental identification of G4 sites in chromatin remains challenging. Here, we report a multifunctional small-molecule ligand, cNDI-CuGGHE, that integrates selective G4 recognition and copper-mediated DNA cleavage within a single molecular scaffold. Spectroscopic analysis showed that cNDI-CuGGHE preferentially binds to various G4 structures through terminal stacking on the G-quartet plane with binding constants on the order of 10⁶ M⁻¹ and significantly stabilizes G4 DNA compared with duplex DNA. Gel electrophoresis confirmed efficient and structure-dependent DNA cleavage in the presence of sodium ascorbate and hydrogen peroxide. Genomic DNA recovered from a cNDI-CuGGHE-based CUT RUN-like assay in HeLa cells was analyzed by quantitative PCR using primers flanking the G4-forming regions of the c-kit and c-myc promoters. The reduced amplification of G4-spanning regions was consistent with preferential DNA cleavage at G4 sites in chromatin. These results demonstrate the feasibility of using small-molecule ligands as alternatives to antibody–enzyme conjugates for G4-targeted chromatin analysis.

Electrochemical gene sensing methods are gaining attention as diagnostic chips. Here, we review the electrochemically active DNA ligand-based sensing methods. Various DNA ligands have been reported in these studies, among which metal complexes, methylene blue, and ferrocenyl naphthalene diimide (FND) have been studied in detail. DNA probe immobilized electrodes have been created, hybridization reactions on the electrodes with target DNA fragments have been performed, and electrochemical gene detection has been possible using these DNA ligands. An example of the realization of this system is the successful and accurate cancer diagnosis using FND to examine abnormal methylation of the hTERT gene, providing reassurance about the system's reliability. In addition, electrochemical detection of PCR products has been realized using the current decrease due to the double-stranded DNA binding of methylene blue although it is a signal-off system. A naphthalene diimide derivative with ferrocene and β-CD, FNC, increased the current upon double-stranded DNA binding. Using these FNCs, the detection of PCR products in a homogeneous system was realized. Electrochemical qPCR was realized with these ligands. Since FNDs also bind strongly to tetraplex or G-quadruplex (G4) DNA, we succeeded in electrochemically detecting telomerase activity, which is known as a cancer marker, using FNDs to detect the amount of telomeric DNA elongation, which is its substrate, as the amount of G4 DNA. This technique has realized compassionate cancer diagnosis from oral swab fluid. It is known that G4 is also present in viral genome RNA, and a viral testing method using G4 is expected to be a potential alternative to PCR. The first example was the electrochemical detection of novel coronaviruses using incFND as an RNA G4 ligand.

A biotinyl cyclic naphthalene diimide (biotinyl cNDI) (1), in which biotin is introduced on the cyclic linker chain of cNDI with high G-quadruplex (G4) specificity, was synthesized. 1 was used for binding analysis to G4 DNAs such as c-myc, c-kit, CEGF, or TA-core. The results showed that 1 bind to G4 DNAs with high affinity and, especially, two molecules of 1 bind to c-myc DNA from top and bottom of G4 site at K = 3.9 × 10−6 M−1 without changing the G4 structure. As a pulldown assay, 1 and streptavidin magnetic beads could be used to recover a c-myc DNA or 120-mer DNA fragment having single c-myc sequence. The qPCR results for the 120-meric DNAs showed that more than 50

The amino acid unit carrying cyclic naphthalene diimide (cNDI) was synthesized by linking the amino moiety of cNDI as a G4-specific ligand with the gamma-carboxylic acid moiety of Fmoc-Glu. cNDI-peptide-dimer, 1 was synthesized by peptide synthesizer using this unit. 1 exhibited an extremely strong binding constant of the order of 108 M-1 with telomeric G4 dimer, probably due to cooperative stacking of the its two G4 planes. It also showed an IC50 at the nM level by the TRAP assay. The results obtained here are expected to enable the design of peptide ligands with multiple cNDI sites targeting the G4 cluster by using the amino acid units of cNDI. Cyclic naphthalene diimide-peptide-dimer 1 showed strong telomerase inhibitory activity with a very strong binding constant on the order of 108 M-1 and an IC50 at the nM level in the TRAP assay. Comparison of the interaction with derivatives of 1 suggested that 1 result was probably due to cooperative stacking of the two G4 planes of telomere G4 dimer. 1 is expected to be a new type of telomerase activity inhibitor and ALT inhibitor. image

Cyclic anthraquinone derivatives (cAQs), which link two side chains of 1,5-disubstituted anthraquinone as a threading DNA intercalator, have been developed as G-quartet (G4) DNA-specific ligands. Among the cAQs, cAQ-mBen linked through the 1,3-position of benzene had the strongest affinity for G4 recognition and stabilization in vitro and was confirmed to bind to the G4 structure in vivo, selectively inhibiting cancer cell proliferation in correlation with telomerase expression levels and triggering cell apoptosis. RNA-sequencing analysis further indicated that differentially expressed genes regulated by cAQ-mBen were profiled with more potential quadruplex-forming sequences. In the treatment of the tumor-bearing mouse model, cAQ-mBen could effectively reduce tumor tissue and had less adverse effects on healthy tissue. These results suggest that cAQ-mBen can be a potential cancer therapeutic agent as a G4 binder.

To develop an electrochemical detection method for COVID-19, we have investigated the interaction between RG-1, a characteristic parallel four-strand RNA component of the COVID-19 genome, and cyclic ferrocenylnaphthalene diimides carrying different linker lengths 1 and 2, which bind to RG-1 more strongly than cyclic naphthalene diimide 3 that lacks a ferrocene moiety. In particular, the binding affinity of 1 having short one showed the highest binding affinity to RG-1, and the redox current of 1 increased in the presence of RG-1 using a glassy carbon electrode. This increasing current of 1 in the presence of c-myc as a parallel G4 DNA or single-stranded RNA was lower than that in the presence of RG-1. Several RG-1 was electrochemically detected at the nanomolar level by 1.

In order to improve the survival rate of oral squamous cell carcinoma (OSCC) patients, a reliable diagnostic method for early OSCC detection is required that is minimally invasive, less burdensome to the patient, and has high sensitivity and specificity. Therefore, we performed the detection of abnormal methylation at three locations in the hTERT promoter region of oral exfoliated cells by employing the ferrocenylnaphthalene diimide (FND)-based electrochemical hybridization assay (FND-EHA) using three types of DNA probe-immobilized electrodes. We also performed liquid cytology using oral exfoliated cells and compared these obtained data to evaluate whether FND- EHA can be used as an OSCC screening system. The results showed a good correlation between this method and conventional OSCC screening, and cytology. In addition, FND-EHA was also able to determine samples that had been ambiguously determined by liquid cytology. This indicates that FND-EHA may be useful as an OSCC screening system.

The cover image is based on the Research Article Electrochemical Method for G-quartet RNA Detection of COVID-19 Based on Cyclic Ferrocenylnaphthalene Diimide by Shuma Kaneyoshi et al., https://doi.org/10.1002/elan.202200427.

Novel cyclic naphthalene diimides, 8 and 12, containing ferrocene in the cyclic linker were synthesized as G quartet (G4) specific electrochemical ligands via the reaction of 1,1 & PRIME;-ferrocenedipropanoic acid and the terminal amine moieties of naphthalene diimides with varying linker lengths. The redox potentials of 8 and 12 were ca. 0.2 V (vs. Ag/AgCl), and the background current in an electrolyte was successfully suppressed. Both 8 and 12 bound to TA-core, representing human telomere G4, with K = 4.4 and 38 x 10(5) M-1, respectively. The current response of 12 to an electrode immobilized with G4 was the highest among the acyclic derivatives, suggesting its potential application in electrochemical telomerase assays.

Potassium-sensing oligonucleotide, PSO, a conjugate of a quadruplex structure-forming oligonucleotide with a peptide incorporating a Förster Resonance Energy Transfer (FRET) chromophore pair, has been developed for fluorescent detection of potassium ion (K+) in aqueous medium. PSO 1 could be introduced into cells for real-time imaging of cytoplasmic K+ concentrations. To perform fluorescent imaging of K+ on the cell surface, we synthesized twelve PSO derivatives with different types of peptide types and lengths, and oligonucleotide sequences including thrombin-binding aptamer (TBA) sequences with FAM and TAMRA as a FRET chromophore pair, and evaluated their performance. 1 was shown to respond selectively to K+, not to most ions present in vivo, and to show reciprocal fluorescence changes in response to K+ concentration. For the peptide chains and oligonucleotide sequences examined in this study, the PSO derivatives had Kd values for K+ in the range of 5–30 mM. All PSO derivatives showed high K+ selectivity even in the presence of excess Na+. The PSO derivatives were successfully localized to the cell surface by biotinylated concanavalin A (ConA) or sulfo-NHS-biotin via streptavidin (StAv). Fluorescence imaging of extracellular K+ upon addition of apoptosis inducers was successfully achieved by 1 localized to the cell surface.

Telomerase is an enzyme that extends telomere sequences to the end of chromosome, and is well known to be overexpressed in more than 80% of cancer cells. Therefore, telomerase is expected as a target for developing novel anticancer drug. As the telomeric repeat sequences form characteristic G-quadruplex (G4) DNA structure, we have investigated the effects of novel compounds derived from G4-binding molecules on the tumor cells. In this study, we investigated the specificity of G4 DNA binding and the anticancer activity of cyclic naphthalene diimide derivatives (cNDIs).

The human telomere region is known to contain guanine-rich repeats and form a guanine-quadruplex (G4) structure. As telomeres play a role in the regulation of cancer progression, ligands that specifically bind and stabilize G4 have potential therapeutic applications. However, as the human telomere sequence can form G4 with various topologies due to direct interaction by ligands and indirect interaction by the solution environment, it is of great interest to study the topology-dependent control of replication by ligands. In the present study, a DNA replication assay of a template with a human telomere G4 sequence in the presence of various ligands was performed. Cyclic naphthalene diimides (cNDI1 and cNDI2) efficiently increased the replication stall of the template DNA at G4 with an anti-parallel topology. This inhibition was stability-dependent and topology-selective, as the replication of templates with hybrid or parallel G4 structures was not affected by the cNDI and cNDI2. Moreover, the G4 ligand fisetin repressed replication with selectivity for anti-parallel and hybrid G4 structures without stabilization. Finally, the method used, referred to as quantitative study of topology-dependent replication (QSTR), was adopted to evaluate the correlation between the replication kinetics and the stability of G4. Compared to previous results obtained using a modified human telomere sequence, the relationship between the stability of G4 and the effect on the topology-dependent replication varied. Our results suggest that native human telomere G4 is more flexible than the modified sequence for interacting with ligands. These findings indicate that the modification of the human telomeric sequence forces G4 to rigidly form a specific structure of G4, which can restrict the change in topology-dependent replication by some ligands.

Newly synthesized naphthalene diimide carrying two β-cyclodextrins (NDI-β-CyDs) showed improved specificity for the parallel G-quadruplex structure alongside the hybrid G-quadruplex structure. Specifically, the highest binding affinity of NDI-β-CyDs for the telomere RNA G-quadruplex was observed. The binding simulation indicated that β-cyclodextrins might be available for loop nucleobase inclusion under its complex.

Porphyromonas gingivalis, known as an anaerobic gram-negative bacteria, is associated with the progress of a periodontitis. Since gingipain (gp), a specific protease secreted from its bacteria, is its main pathogenicity, gp has been utilized as a marker of the periodontitis. Here, an electrochemical protease assay using a ferrocenylpeptide probes was applied to the detection of gp activity; the probe was cleaved with its protease activity and current peak decreased by the released ferrocene part to balk solution. A series of ferrocenylpeptides as specific substrates of Arg-gp (Rgp) and Lys-gp (Kgp) was synthesized: FRC and FKC having cysteine residue; FRSS and FKSS having lipoic acid; FRpra and FKpra having propargylglycine; FRDE and FRDK having cysteine and D-type amino acid. The electron transfer rate constant of all of ferrocenylpeptide immobilized on a gold electrode were estimated by Lavirons analysis. The obtained electron transfer rate constant were 400-3500 s(-1), where fastest one of 3500 s(-1) was obtained from FRC immobilized electrode. Peak currents of these electrodes were decreased after the treatment of a sample containing Porphyromonas gingivalis The detection limits of Porphyromonas gingivalis were 1.0 x 10(7) , 5.0 x 10(6) , 3.3 x 10(6) , 5.0 x 10(6) , and 5.0 x 10(6) cells, for FRPra- FKPra-, FRG, FRDE-, and FRDK-immobilized electrodes, respectively. FRDE- and FRDK-immobilized electrodes were applied for patients suffering from periodontitis. It was successfully classified between the patients and healthy peoples using the FRDK-immobilized electrode. These results suggest that gp activity is connected with a clinical condition of periodontitis, and it is expected to be applied to a periodontal disease screening.

Interaction of cyclic naphthalene diimide derivatives (cNDIs), 1-4, with TA-core and c-myc as G-quartet (G4) DNA was studied under dilute or molecular crowding condition. Binding study for TA-core based on an isothermal titration calorimetry showed that 1-4 has 10(6) M-1 order of binding affinity with the following order: 1 4 > 2 > 3 under both conditions. Meting temperature (T-m) of TA-core obtained from the temperature dependence of circular dichroism spectra shows that TA-core was most stabilized by 4, which is in agreement with the result of PCR stop assay and the stabilization effect for 1-3 was correlated with their binding affinity under dilute condition. 3 showed specific growth inhibition of cancer cell line Ca9-22 at <0.03 mu M of IC50, with no inhibitory effect against normal bone marrow cells. 3, which has highest value of Delta H/Delta G, shows the highest inhibition ability for Ca9-22, carrying a highest expression level of telomerase mRNA.

We present a safe approach of minimally invasive cardiac surgery (MICS) through thoracotomy using continuous retrograde cardioplegia through anterior thoracotomy. Continuous retrograde cardioplegia allows excellent continuous homogenous cooling of the heart during the ischemic period. Anterior thoracotomy facilitates cannulation of ascending aorta, and allows our all manipulations using the fingertips without the aid of a knot pusher or long-shafted surgical instruments.

Ligands that bind to and stabilize guaninequadruplex (G4) structures to regulate DNA replication have therapeutic potential for cancer and neurodegenerative diseases. Because there are several G4 topologies, ligands that bind to their specific types may have the ability to preferentially regulate the replication of only certain genes. Here, we demonstrated that binding ligands stalled the replication of template DNA at G4, depending on different topologies. For example, naphthalene diimide derivatives bound to the G-quartet of G4 with an additional interaction between the ligand and the loop region of a hybrid G4 type from human telomeres, which efficiently repressed the replication of the G4. Thus, these inhibitory effects were not only stability-dependent but also topology-selective based on the manner in which G4 structures interacted with G4 ligands. Our original method, referred to as a quantitative study of topology-dependent replication (QSTR), was developed to evaluate correlations between replication rate and G4 stability. QSTR enabled the systematic categorization of ligands based on topology-dependent binding. It also demonstrated accuracy in determining quantitatively how G4 ligands control the intermediate state of replication and the kinetics of G4 unwinding. Hence, the QSTR index would facilitate the design of new drugs capable of controlling the topology-dependent regulation of gene expression.