Binding Of Probe Research Articles

Label-free, ultra-low detection limit DNA biosensor using high quality optical microcavity functionalized by DNA tetrahedral nanostructure probes.

This study proposes and demonstrates a novel label-free DNA biosensor using high quality optical microcavity functionalized by 3D DNA nanostructure probes. To achieve ultra-low limit DNA biosensing, optical sensing interface of the hollow-core, thin wall-thickness microcavity was functionalized by self-assembled DNA tetrahedral nanostructure (DTN) probes with size of 17 bp and length of ∼5.8 nm. High efficiency binding of the DTN probes with the optical sensor interface and the target ssDNA are achieved. Whispering gallery mode (WGM) spectra with high-Q factor of >107 are excited and traced for DNA detection inside the microfluidic channel of the microcavity, with a small sample volume of nL. Incorporation of nanoscale DTN probes onto surface of the optical microcavity makes it an effective way for increasing efficient probe density and eliminating entanglement between DNA probes, thus ∼1000 times lower detection limit is achieved as compared to using 1D ssDNA probes. Due to its desirable merits of label-free, ultra-low LoD, real time and compact size, the proposed DNA biosensor has broad application prospects in bioengineering and medical diagnosis.

Evaluation of ω-alkynyl-labeled linoleic and arachidonic acids as substrates for recombinant lipoxygenase pathway enzymes

ω-Alkynyl-fatty acids can be used as probes for covalent binding to intracellular macromolecules. To inform future in vivo studies, we determined the rates of reaction of ω-alkynyl-labeled linoleate with recombinant enzymes of the skin 12R-lipoxygenase (12R-LOX) pathway involved in epidermal barrier formation (12R-LOX, epidermal lipoxygenase-3 (eLOX3), and SDR9C7). We also examined the reactivity of ω-alkynyl-arachidonic acid with representative lipoxygenase enzymes employing either “carboxyl end-first” substrate binding (5S-LOX) or “tail-first” (platelet-type 12S-LOX). ω-Alkynyl-linoleic acid was oxygenated by 12R-LOX at 62 ± 9 % of the rate compared to linoleic acid, the alkynyl-9R-HPODE product was isomerized by eLOX3 at only 43 ± 1 % of the natural substrate, whereas its epoxy alcohol product was converted to epoxy ketone linoleic by an NADH-dependent dehydrogenase (SDR9C7) with 91 ± 1 % efficiency. The results suggest the optimal approach will be application of the 12R-LOX/eLOX3-derived epoxyalcohol, which should be most efficiently incorporated into the pathway and allow subsequent analysis of covalent binding to epidermal proteins. Regarding the orientation of substrate binding in LOX catalysis, our results and previous reports suggest the ω-alkynyl group has a stronger inhibitory effect on tail-first binding, as might be expected. Beyond slowing the reaction, however, we found that the tail-first binding and transformation of ω-alkynyl-arachidonic acid by platelet-type 12S-LOX results in almost complete enzyme inactivation, possibly due to reactive intermediates blocking the enzyme active site. Overall, the results reinforce the conclusion that ω-alkynyl-fatty acids are suitable for selected applications after appropriate reactivity is established.

Exploring magnetic capture to improve the detection of human norovirus in strawberries

AbstractHuman norovirus is a leading cause of foodborne illness worldwide, and its detection in foods remains a challenge due to the difficulty of eluting and enriching trace amounts of viral particles. In this study, we developed porcine gastric mucin–labeled magnetic capture probes and optimized the conditions for their binding with norovirus, elution of viral particles, and RNA isolation based on the previous research. We evaluated the magnetic capture‐based pretreatment method in artificially contaminated strawberry samples and compared it with the conventional polyethylene glycol precipitation–based method using reverse transcriptional‐quantitative PCR (RT‐qPCR). The results revealed that Tris·HCl (pH 9.5) was the optimal buffer for the binding of magnetic probes with viral particles, and RNA recovery rate obtained by heating‐based release was significantly higher than that obtained by a commercial kit, with only active virus being captured. This buffer was similar to the RT‐qPCR buffer, which facilitated the sensitivity of RT‐qPCR. Furthermore, the buffer was an effective solution for eluting viral particles from foods in previous research and was optimized to ensure maximum elution and no inhibitory effect on viral capture. We explored multiple factors or steps to ensure that the sensitivity of our method was three orders of magnitude higher than that of the conventional method. Our findings suggest that magnetic capture‐based detection has great potential for the sensitive detection of human norovirus.

Nanoparticle-Coupled Single-Molecule Kinetic Fingerprinting for Enzymatic Activity Detection.

The sensitive and accurate detection of biomarkers plays an important role in clinical diagnosis and drug discovery. Currently, amplification-based methods for biomarker detection are widely explored. However, the key challenges of these methods are limited reproducibility and high background noise. To overcome these limitations, we develop a robust plasmonic nanoparticle-coupled single-molecule kinetic fingerprinting (PNP-SMKF) method to achieve ultrasensitive detection of protein kinase A (PKA). Transient binding of a short fluorescent probe with the genuine target produces a distinct kinetic signature that is completely different from that of the background signal, allowing us to recognize PKA sensitively. Importantly, integrating a plasmonic nanoparticle efficiently breaks the concentration limit of the imager strand for single-molecule imaging, thus achieving a much faster imaging speed. A limit of detection (LOD) of as low as 0.0005 U/mL is readily realized. This method holds great potential as a versatile platform for enzyme detection and inhibitor screening in the future.

Biophysical and in-silico studies on the structure-function relationship of Brugia malayi protein disulfide isomerase

Human Lymphatic filariasis is caused by parasitic nematodes Wuchereria bancrofti, Brugia malayi, and Brugia timori. Protein disulfide isomerase (PDI), a redox-active enzyme, helps to form and isomerize the disulfide bonds, thereby acting as a chaperone. Such activity is essential for activating many essential enzymes and functional proteins. Brugia malayi protein disulfide isomerase (BmPDI) is crucial for parasite survival and an important drug target. Here, we used a combination of spectroscopic and computational analysis to study the structural and functional changes in the BmPDI during unfolding. Tryptophan fluorescence data revealed two well-separated transitions during the unfolding process, suggesting that the unfolding of the BmPDI is non-cooperative. The binding of the fluorescence probe 8-anilino-1-naphthalene sulfonic acid dye (ANS) validated the results obtained by the pH unfolding. The dynamics of molecular simulation performed at different pH conditions revealed the structural basis of BmPDI unfolding. Detailed analysis suggested that under different pH, both the global structure and the conformational dynamics of the active site residues were differentially altered. Our multiparametric study reveals the differential dynamics and collective motions of BmPDI unfolding, providing insights into its structure-function relationship. Communicated by Ramaswamy H. Sarma

European Society of Toxicologic Pathology (Pathology 2.0 Molecular Pathology Special Interest Group): Review of In Situ Hybridization Techniques for Drug Research and Development.

In situ hybridization (ISH) is used for the localization of specific nucleic acid sequences in cells or tissues by complementary binding of a nucleotide probe to a specific target nucleic acid sequence. In the last years, the specificity and sensitivity of ISH assays were improved by innovative techniques like synthetic nucleic acids and tandem oligonucleotide probes combined with signal amplification methods like branched DNA, hybridization chain reaction and tyramide signal amplification. These improvements increased the application spectrum for ISH on formalin-fixed paraffin-embedded tissues. ISH is a powerful tool to investigate DNA, mRNA transcripts, regulatory noncoding RNA, and therapeutic oligonucleotides. ISH can be used to obtain spatial information of a cell type, subcellular localization, or expression levels of targets. Since immunohistochemistry and ISH share similar workflows, their combination can address simultaneous transcriptomics and proteomics questions. The goal of this review paper is to revisit the current state of the scientific approaches in ISH and its application in drug research and development.

NTSR1 glycosylation and MMP dependent cleavage generate three distinct forms of the protein

NTSR1 abnormal expression by cancer cells makes it a strategic target for antitumoral therapies, such as compounds that use NTSR1 binding probes to deliver cytotoxic agents to tumor cells. Success of these therapies relies on NTSR1 protein availability and accessibility; therefore, understanding the protein’s biology is crucial. We studied NTSR1 protein in exogenously and endogenously expressing non-tumoral and tumoral cells. We found NTSR1 to be expressed as three distinct protein forms: the NTSR1-high form, a glycosylated protein; the NTSR1-low form, a N-terminally cleaved and de-glycosylated protein; and the NTSR1-LP protein with the MW size predicted by its NTSR1 amino acid sequence. We show that the NTSR1-high form is cleaved by MMPs to generate the NTSR1-low form, a process that is promoted by the Neurotensin (NTS) ligand. In addition, NTS induced the internalization of plasma membrane localized NTSR1 and degradation of NTSR1-low form via the proteasome. Importantly, we found NTSR1-low form to be the most abundant form in the tumoral cells and in PDAC Patient Derived Xenograft, demonstrating its physiopathological relevance. Altogether, our work provides important technical and experimental tools as well as new crucial insights into NTSR1 protein biology that are required to develop clinically relevant NTSR1 targeting anti-tumoral therapies.

Advances in Activity‐Based Protein Profiling of Functional Tyrosines in Proteomes

AbstractActivity‐based protein profiling (ABPP) is a chemical proteomic method for investigating functional states of proteins in native biological settings. By quantifying changes in probe binding states of active and regulatory protein sites, ABPP reveals functional information on protein regulation and can be configured in competitive settings to determine global selectivity profiles of tool compounds and drugs in lysates, cells, and animals. Chemical probes used for ABPP analyses can target protein families with conserved enzymatic or structural features or can broadly profile the proteome using electrophiles with reactivity towards functional groups on amino acid side chains. The latter approach has provided insights to protein sites involved in allosteric regulation and non‐enzymatic functions. This review introduces quantitative ABPP workflows and discusses electrophilic groups used for ABPP profiling of functional sites in the proteome with an emphasis on tyrosine residues.

Towards a unified pipeline for tissue-scale localization and identification of individual RNA molecules.

Single-molecule fluorescent in situ hybridization (smFISH) is a leading method for gene transcription analysis at the cellular level, yielding information on gene expression levels and spatial RNA distributions. When extended to the tissue or organ level, this information helps guide development of targeted treatments for disease, such as personalized cancer therapies. However, smFISH data is inherently hindered by inhomogeneous background, detector noise, optical aberrations, clustering of RNA transcripts at transcription sites, and the inherently stochastic nature of probe binding.

Understanding cooperativity effects in RNA structure probing experiments.

Small molecule chemical probes, which form covalent bonds with atoms of flexible nucleotides, are commonly used to gain insight into the structure adopted by an RNA. In this work, we use selective 2’ hydroxyl acylation analyzed by primer extension (SHAPE) and nuclear magnetic resonance (NMR) spectroscopy experiments to explore whether the structure of an RNA is modulated by chemical probe binding events. We find that as the concentration of a chemical probe increases, modified nucleotides locally modulate the RNA structure, resulting in the increase or decrease of SHAPE reactivity in surrounding nucleotides. This cooperative effect is dependent on both chemical probe concentration and size. Critically, this effect impacts the accuracy of predicted RNA secondary structures when guided by chemical probing experiments. Altogether, our work highlights the importance of optimizing the conditions of chemical probing experiments to obtain secondary structural information about an RNA.

Peptide nucleic acid and antifouling peptide based biosensor for the non-fouling detection of COVID-19 nucleic acid in saliva

The electrochemical biosensor with outstanding sensitivity and low cost is regarded as a viable alternative to current clinical diagnostic techniques for various disease biomarkers. However, their actual analytical use in complex biological samples is severely hampered due to the biofouling, as they are also highly sensitive to nonspecific adsorption on the sensing interfaces. Herein, we have constructed a non-fouling electrochemical biosensor based on antifouling peptides and the electroneutral peptide nucleic acid (PNA), which was used as the recognizing probe for the specific binding of the viral RNA of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Different from the negatively charged DNA probes that will normally weaken the biosensors’ antifouling capabilities owing to the charge attraction of positively charged biomolecules, the neutral PNA probe will generate no side-effects on the biosensor. The biosensor demonstrated remarkable sensitivity in detecting SARS-CoV-2 viral RNA, possessing a broad linear range (1.0 fM - 1.0 nM) and a detection limit down to 0.38 fM. Furthermore, the sensing performance of the constructed electrochemical biosensor in human saliva was nearly similar to that in pure buffer, indicating satisfying antifouling capability. The combination of PNA probes with antifouling peptides offered a new strategy for the development of non-fouling sensing systems capable of assaying trace disease biomarkers in complicated biological media.

Catalysis and inhibition of ester hydrolysis by encapsulation in micelles derived from designer surfactant TPGS-750-M

The designer surfactant TPGS-750-M is an important technology for green and sustainable chemical synthesis that facilitates a wide variety of organic transformations in water. Despite its growing importance in organic synthesis, many basic aspects of binding and reactivity within TPGS-750-M micelles remain poorly understood. In this work, the impact of TPGS-750-M on the rate of p-nitrophenyl ester hydrolysis kinetics was investigated to probe binding strength within TPGS-750-M micelles and the chemical environment of the micelle interior. We found that the hydrolysis rates of p-nitrophenyl acetate and p-nitrophenyl hexanoate were slowed by the presence of TPGS-750-M by up to 92%, consistent with a hydrophobic encapsulation environment within the micelle interior. The hydrolysis rates of p-nitrophenyl decanoate and p-nitrophenyl hydroxycinnamate were accelerated up to 74-fold by the presence of TPGS-750-M, but only at low surfactant concentrations. We propose that these rate enhancements are due to mixed micelle formation between ester and TPGS-750-M, which positions the reactive ester group within the hydrophilic region of the micelle. This study suggests that there are two distinct chemical environments present in the interior of micelles derived from TPGS-750-M, and that it is possible for very hydrophobic substrates to interact with the polar, hydrophilic region of the micelle. This work also demonstrates that increased hydrophobicity leads to stronger substrate binding within TPGS-750-M micelles.

Rapid Assessment of SARS-CoV-2 Variant-Associated Mutations in Wastewater Using Real-Time RT-PCR.

Within months of the COVID-19 pandemic being declared on March 20, 2020, novel, more infectious variants of SARS-CoV-2 began to be detected in geospatially distinct regions of the world. With international travel being a lead cause of spread of the disease, the importance of rapidly identifying variants entering a country is critical. In this study, we utilized wastewater-based epidemiology (WBE) to monitor the presence of variants in wastewater generated in managed COVID-19 quarantine facilities for international air passengers entering the United Kingdom. Specifically, we developed multiplex reverse transcription quantitative PCR (RT-qPCR) assays for the identification of defining mutations associated with Beta (K417N), Gamma (K417T), Delta (156/157DEL), and Kappa (E154K) variants which were globally prevalent at the time of sampling (April to July 2021). The assays sporadically detected mutations associated with the Beta, Gamma, and Kappa variants in 0.7%, 2.3%, and 0.4% of all samples, respectively. The Delta variant was identified in 13.3% of samples, with peak detection rates and concentrations observed in May 2021 (24%), concurrent with its emergence in the United Kingdom. The RT-qPCR results correlated well with those from sequencing, suggesting that PCR-based detection is a good predictor for variant presence; although, inadequate probe binding may lead to false positive or negative results. Our findings suggest that WBE coupled with RT-qPCR may be used as a rapid, initial assessment to identify emerging variants at international borders and mass quarantining facilities. IMPORTANCE With the global spread of COVID-19, it is essential to identify emerging variants which may be more harmful or able to escape vaccines rapidly. To date, the gold standard to assess variants circulating in communities has been the sequencing of the S gene or the whole genome of SARS-CoV-2; however, that approach is time-consuming and expensive. In this study, we developed two duplex RT-qPCR assays to detect and quantify defining mutations associated with the Beta, Gamma, Delta, and Kappa variants. The assays were validated using RNA extracts derived from wastewater samples taken at quarantine facilities. The results showed good correlation with the results of sequencing and demonstrated the emergence of the Delta variant in the United Kingdom in May 2021. The assays developed here enable the assessment of variant-specific mutations within 2 h after the RNA extract was generated which is essential for outbreak rapid response.

Molecular basis of structural stability of Irisin: A combined molecular dynamics simulation and in vitro studies for Urea-induced denaturation

Irisin is a recently discovered myokine having important role in adipose tissue metabolism, neuronal functioning as well as bone metabolism. In this study, urea-induced denaturation was performed to determine the conformational stability and equilibrium folding intermediate of irisin. Understanding of the folding/unfolding pathway is fundamental to study the structure and stability of proteins. Herein, the urea-induced conformational changes in irisin were monitored employing spectroscopic techniques as well as all-atom molecular dynamics (MD) simulations. It was revealed that process of irisin unfolding is biphasic, i.e., native state (N) ↔ intermediate state (I) ↔ denatured state (D) with I state populated around 2.5 M urea. The stabilized intermediate state at 2.5 M urea was confirmed through binding of the fluorescence probe, 1-anilino-8-naphthalene sulfonate (ANS). Further, 250 ns MD simulation of irisin in urea was carried out to have detailed mechanistic insight. The assessment of structural parameters, free energy landscapes and native contacts indicated the presence of intermediate state during unfolding of irisin, which was characterised as molten globule state. The results of MD simulation showed a strong correlation with the in vitro observations establishing the occurrence of the intermediate population during the unfolding of irisin induced by urea.

Binding interactions of hydrophobically-modified flavonols with β-glucosidase: fluorescence spectroscopy and molecular modelling study.

Natural flavonoids are capable of inhibiting glucosidase activity, so they can be used for treating diabetes mellitus and hypertension. However, molecular-level details of their interactions with glucosidase enzymes remain poorly understood. This paper describes the synthesis and spectral characterization of a series of fluorescent flavonols and their interaction with the β-glucosidase enzyme. To tune flavonol-enzyme interaction modes and affinity, we introduced different polar halogen-containing groups or bulky aromatic/alkyl substituents in the peripheral 2-aryl ring of a flavonol moiety. Using fluorescence spectroscopy methods in combination with molecular docking and molecular dynamics simulations, we examined the binding affinity and identified probe binding patterns, which are critical for steric blockage of the key catalytic residues of the enzyme. Using a fluorescent assay, we demonstrated that the binding of flavonol 2e to β-glucosidase decreased its enzymatic activity up to 3.5 times. In addition, our molecular docking and all-atom molecular dynamics simulations suggest that the probe binding is driven by hydrophobic interactions with aromatic Trp and Tyr residues within the catalytic glycone binding pockets of β-glucosidase. Our study provides a new insight into structure-property relations for flavonol-protein interactions, which govern their enzyme binding, and outlines a framework for a rational design of new flavonol-based potent inhibitors for β-glucosidases.

Architecture of dual emissive three-dimensional nanostructure composites containing graphitic 2D sheets and iron oxide nanoparticles: detection of short single-stranded DNA sequences.

Here, we report an effect driven by repetitive heating and cooling; as a result, 2D and 1D nanomaterials are parallelly produced locally in a single reactor from the same precursors. Afterward, more repetitive heating and cooling induced the self-folding approach of a 2D nanomaterial with a 1D nanomaterial, giving them a self-assembled biconcave disk-shaped 3D nanostructure. The microscopy and spectroscopy studies reveal that the nanostructure has a diameter of nearly 200 nm and is composed of Fe, C, O and incorporated N and P. This 3D nanostructure composite shows red-shifted dual emission (430 nm and 500 nm) at two different excitations (350 nm and 450 nm), accompanied by a rare large Stokes shift (LSS), and it was employed in the detection of targeted short single-stranded DNA sequences (ssDNA). Upon the addition of target DNA, the specific binding of 3D nanostructure probes with the target triggers variations (off/on) of two signals, and by considering the decreased emission (fluorescence quenching) at 500 nm, we can detect the target ssDNA at the single-molecule level. The change of fluorescence intensity and the concentration of complementary target ssDNA sequences show a better linear relationship than a single emission-based probe, and the limit of detection (LOD) was as low as 0.47 nM.

Predicting small molecule binding pockets on diacylglycerol kinases using chemoproteomics and AlphaFold.

Diacylglycerol kinases (DGKs) are metabolic kinases involved in regulating cellular levels of diacylglycerol and phosphatidic lipid messengers. The development of selective inhibitors for individual DGKs would benefit from discovery of protein pockets available for inhibitor binding in cellular environments. Here we utilized a sulfonyl-triazole probe (TH211) bearing a DGK fragment ligand for covalent binding to tyrosine and lysine sites on DGKs in cells that map to predicted small molecule binding pockets in AlphaFold structures. We apply this chemoproteomics-AlphaFold approach to evaluate probe binding of DGK chimera proteins engineered to exchange regulatory C1 domains between DGK subtypes (DGKα and DGKζ). Specifically, we discovered loss of TH211 binding to a predicted pocket in the catalytic domain when C1 domains on DGKα were exchanged that correlated with impaired biochemical activity as measured by a DAG phosphorylation assay. Collectively, we provide a family-wide assessment of accessible sites for covalent targeting that combined with AlphaFold revealed predicted small molecule binding pockets for guiding future inhibitor development of the DGK superfamily.

Fluorescence probing and molecular docking analysis of the binding interaction of bovine serum albumin (BSA) with the polarity probe AICCN.

In this work, the fluorescent probe 2-amino-4-(1H-indol-3-yl)-4H-chromene-3-carbonitrile (AICCN) has been used to evaluate its potential as a prospective polarity probe. From detailed fluorescence studies of the probe, it could be shown that AICCN can indeed function as an effective polarity probe. The calculated dipole moments of AICCN in both the ground state and excited state in various solvents lend support to the steady state fluorescence results. It was also shown that AICCN can be used to probe the micropolarity of micelles and can be used successfully for the determination of CMC of the surfactants. The binding process of the probe AICCN to BSA has been followed by plotting the binding isotherms and Scatchard Plots. The time-resolved fluorescence data indicate that the preferred binding site of AICCN in BSA lies close to the buried Trp residue Trp-213 in Domain II. This contention is further supported by the molecular docking studies. The interaction study of the probe AICCN with proteins is relevant for future use of AICCN as a hydrophobic drug. Information was also obtained about the effect of probe binding on the serum albumin structure, which may be correlated to its physiological activity. Thus, the probe AICCN can serve not only as a good reporter of polarity of the microenvironment in biological systems but also as an efficient fluorophore to monitor conformational changes in proteins in future.

Validation and implementation of an orthopoxvirus qualitative real-time PCR for the diagnosis of monkeypox in the clinical laboratory.

The globally concerning outbreak of monkeypox virus (MPXV) in non-endemic countries in 2022 warranted an increased capacity of diagnostic clinical testing. In this study, we detail the Johns Hopkins analytical validation of a qualitative orthopoxvirus real-time PCR and characterize its analytical performance for MPXV testing. We also describe our first month of MPXV clinical laboratory diagnosis, including assay utilization and positivity. The analytical performance of a previously characterized orthopoxvirus assay that targets the DNA polymerase gene of non-variola orthopoxviruses was determined. In silico inclusivity analysis was performed for the primer and probe sequences. The limit of detection, reproducibility, agreement, and specificity were evaluated as well as target stability at room temperature. Clinical chart reviews were performed for patients who received testing within the first month of offering the assay for diagnosis. Alignment of the primer and probe binding regions of 151 sequenced genomes of MPXV from 2022 showed Sequence homology of 100%. The assay was able to detect MPXV at a concentration of at least 100 copies/mL and showed no cross reactivity with other organisms tested. Targets were stable for at least one week at room temperature. During the first month of implementation, a total of 33 patients were tested, 11 of whom were positive and presented mainly with rash in the genital region. Increased availability of MPXV testing is essential with the progressive increase in the number of cases. The non-variola orthopoxvirus assay is a sensitive and reproducible method for diagnosing monkeypox during the current surge in cases.

Positional Isomeric Symmetric Dipodal Receptors Dangled with Rotatable Binding Scaffolds: Fluorescent Sensing of Silver Ions and Sequential Detection of l-Histidine and Their Multifarious Applications

Three simple dipodal artificial acyclic symmetric receptors, SDO, SDM, and SDP, driven by positional isomerism based on xylelene scaffolds were designed, synthesized, and characterized by 1H NMR, 13C NMR, and mass spectroscopy techniques. Probes SDO, SDM, and SDP demonstrated selective detection of Ag+ metal ions and amino acid l-histidine in a DMSO-H2O solution (1:1 v/v, HEPES 50 mM, pH = 7.4). The detection of Ag+ metal ions occurred in three ways: (i) inhibition of the photoinduced electron-transfer (PET) process, (ii) blueshifted fluorescence enhancement via the intramolecular charge-transfer (ICT) process, and (iii) restricted rotation of the dangling benzylic scaffold following coordination with a Ag+ metal ion. Job's plot analysis and quantum yields confirm the binding of probes to Ag+ in 1:1, 1:2, and 1:2 ratios with LODs and LOQs found to be 1.3 μM and 3.19 × 10-7 M, 6.40 × 10-7 and 2.44 × 10 -6 M, and 9.76 × 10-7 and 21.01 × 10-7 M, respectively. 1H NMR titration, HRMS, ESI-TOF, IR analysis, and theoretical DFT investigations were also used to establish the binding stoichiometry. Furthermore, the probes were utilized for the detection of Ag+ ions in water samples, food samples, soil analysis, and bacterial imaging in Escherichia coli cells and a molecular logic gate was constructed.