Probe For Detection Research Articles

A highly sensitive and selective fluorescent “on–off-on” peptide-based probe for sequential detection of Hg2+ and S2− ions: Applications in living cells and zebrafish imaging

Mercury ions (Hg2+) and sulfur ions (S2−), have caused serious harm to the ecological environment and human health as two kinds of highly toxic pollutants widely used. Therefore, the visual quantitative determination of Hg2+ and S2− is of great significance in the field of environmental monitoring and medical therapy. In this study, a novel fluorescent “on–off-on” peptide-based probe DNC was designed and synthesized using dipeptide (Asn-Cys-NH2) as the raw material via solid phase peptide synthesis (SPPS) technology with Fmoc chemistry. DNC displayed high selectivity in the recognition of Hg2+, and formed non-fluorescence complex (DNC-Hg2+) through 2:1 binding mode. Notably, DNC-Hg2+ complex generated in situ was used as relay response probe for highly selective sequential detection of S2− through reversible formation-separation. DNC achieved highly sensitive detection of Hg2+ and S2− with the detection limits (LODs) of 8.4 nM and 5.5 nM, respectively. Meanwhile, DNC demonstrated feasibility for Hg2+ and S2− detections in two water samples, and the considerable recovery rate was obtained. More importantly, DNC showed excellent water solubility and low toxicity, and was successfully used for consecutive discerning Hg2+ and S2− in test strips, living cells and zebrafish larvae. As an effective visual analysis method in the field, smartphone RGB Color Picker APP realized semi-quantitative detections of Hg2+ and S2− without the need for complicated device.

Multiplexed in situ RNA imaging by combFISH.

Multiplexed in situ RNA imaging offers new opportunities for gene expression profiling by providing high-throughput spatial information. In this work, we present a cyclic combinatorial fluorescent in situ hybridization (combFISH) assay to achieve multiplexed detection of RNA in cell cultures and tissues. Specifically, multiplexing is achieved through cyclic interrogation of barcode sequences on the rolling circle amplicons generated from the padlock probe assay by using sets of combinatorial detection probes. Theoretically, combFISH can detect 64 genes in three hybridization cycles by combinatorial barcoding using 12 fluorescently labeled detection probes. Our method eliminates sequencing-by-ligation (SBL) chemistry in the in situ sequencing protocol and directly uses RNA as targets for ligation, making it more straightforward. We showed that our method works in fresh-frozen and formalin-fixed paraffin-embedded tissue sections. With its straightforward protocols, we expect our method to be adopted by the scientific community and extended to clinical settings.

Old drug, new use: The thalidomide-based fluorescent probe for cysteine detection and imaging in living cells

Thalidomide, as a high-profile cereblon (CRBN) ligand, has attracted much attention because of its ability to target protein degradation. In this study, we are committed to developing a new fluorescent probe THD-1 based on thalidomide, aiming at improving the performance of cysteine fluorescent probe in optical properties and biocompatibility. The experimental results showed that THD-1, as a cysteine fluorescent probe, owned the characteristics of obvious colorimetric change, fast response time, good selectivity and high sensitivity. The mechanism of THD-1 sensing cysteine was further verified to ensure its reliability and effectiveness. It was also worth mentioning that THD-1 was successfully applied to the biological imaging of cysteine in living A549 cells, which highlighted its value in practical application. Overall, thalidomide, as a clinically approved drug, not only enriches the fluorescent skeleton library, but also paves a new way for the further development of fluorescent probes.

Detecting the FLJ22447 lncRNA in Ovarian Cancer with Cyclopentane-Modified FIT-PNAs (cpFIT-PNAs).

Ovarian cancer (OC) is one of the most lethal gynecologic cancers that is typically diagnosed at the very late stage of disease progression. Thus, there is an unmet need to develop diagnostic probes for early detection of OC. One approach may rely on RNA as a molecular biomarker. In this regard, FLJ22447 lncRNA is an RNA biomarker that is over-expressed in ovarian cancer (OC) and in cancer-associated fibroblasts (CAFs). CAFs appear early on in OC as they provide a metastatic niche for OC progression. FIT-PNAs (forced intercalation-peptide nucleic acids) are DNA analogs that are designed to fluoresce upon hybridization to their complementary RNA target sequence. In recent studies, we have shown that the introduction of cyclopentane PNAs into FIT-PNAs (cpFIT-PNA) results in superior RNA sensors. Herein, we report the design and synthesis of cpFIT-PNAs for the detection of this RNA biomarker in living OC cells (OVCAR8) and in CAFs. cpFIT-PNA was compared to FIT-PNA and the cell-penetrating peptide (CPP) of choice was either a simple one (four L-lysines) or a CPP with enhanced cellular uptake (CLIP6). The combination of CLIP6 with cpFIT-PNA resulted in a superior sensing of FLJ22447 lncRNA in OVCAR8 cells as well as in CAFs. Moreover, incubation of CLIP6-cpFIT-PNA in OVCAR8 cells leads to a significant decrease (ca. 60%) in FLJ22447 lncRNA levels and in cell viability, highlighting the potential theranostic use of such molecules.

Near-infrared fluorescent probe for sensitive detection and imaging of DNA G4s in living cells

G-quadruplexs (G4s), four-stranded nucleic acid secondary structures, which formed by guanine-rich sequences play a vital role in human biological systems. Studies have shown that the formation of G4s is closely related to tumor development and apoptosis, which is considered as a new target for the development of anti-tumor drugs. Therefore, it is important to develop novel probes for G4s imaging. In this article, we engineered a near-infrared fluorescent probe (TOH) which can be activated by DNA G4s in living cells and tumor. TOH exhibits high selectivity to the structure of DNA G4s with the limit of detection for DNA G4s (Mito-0.5–2) is calculated to be 0.43 nM. Imaging studies of different cell lines revealed that the brighter fluorescence in cancer cell lines than in normal, indicating that DNA G4s maybe highly express in tumor cell lines. Simultaneously, TOH is also introduced into live tumor tissue imaging and found that the fluorescence intensity of tumor is the brightest relative to normal tissue, further validating the high expression of DNA G4s structures in tumor tissue. These features demonstrate TOH not only have the ability to image DNA G4 structures in real time, but also may have tumor diagnostic capabilities.

Highly-efficient selection of interferon gamma-specific aptamers and development of a sensitive fiber-optic evanescent wave aptasensor

Interferon-gamma (IFN-γ) is a critical indicator of the immune response to many infections and diseases. Identifying excellent IFN-γ recognition molecules is of great significance for the early diagnosis and treatment of diseases. Herein, anti-IFN-γ aptamers were selected based on capillary electrophoresis-systematic evolution of ligands by exponential enrichment (CE-SELEX) and their affinities were characterized by the biolayer interferometry (BLI) assay. A rational molecular docking strategy was utilized to predict precisely the binding behavior between aptamer and IFN-γ and remove redundant bases of original sequence. Therefore, aptamer IFNG-8 with the highest affinity can be efficiently identified within only three rounds and the truncated aptamer IFNG-8 T with comparable affinity is eventually obtained. With IFNG-8 T as the detection probe, a fiber-optic evanescent wave aptasensor is constructed for the detection of IFN-γ, which shows a wide linear range from 0.1 to 10000 pM and a low limit of detection (LOD) of 0.0718 pM. The developed aptasensor exhibits high selectivity and can be utilized for IFN-γ detection in human serum samples with good recoveries. In summary, the truncated aptamer can serve as a novel optional transducing element and the developed aptasensor provides an innovative methodology for detection of IFN-γ in trace amount.

Residual Magnetic Field Testing System with Tunneling Magneto-Resistive Arrays for Crack Inspection in Ferromagnetic Pipes.

Ferromagnetic pipes are widely used in the oil and gas industry. They are subject to cracks due to corrosion, pressure, and fatigue. It is significant to detect cracks for the safety of pipes. A residual magnetic field testing (RMFT) system is developed for crack detection in ferromagnetic pipes. Based on this background, a detection probe based on an array of tunneling magneto-resistive (TMR) sensors and permanent magnets is exploited. The probe is able to partially magnetize the pipe wall and collect magnetic signals simultaneously. First, a theoretical analysis of RMFT is presented. The physics principle of RMFT is introduced, and a finite element model is built. In the finite element simulations, the effects of the crack length and depth on the RMFT signal are analyzed, and the signal characteristics are selected to represent the crack size. Next, the validated experiments are conducted to demonstrate the feasibility of the proposed RMFT method in this paper.

Coordinated Solvent Molecules Enable the Excellent Capabilities of Two Zn2+‐Based Complexes in Detecting l‐Arginine via Long‐Lived Luminescence Recovery

AbstractLuminescence metal–organic materials (MOMs) are widely used as probes for detection. However, most of such probes are based on fluorescence and work in either turn‐off or turn‐on mode. In contrast, long‐lived (>10 ms) probes (LLPs) with recovery response to analyte are quite rare. Herein “solvation complex” strategy is used to prepare two new afterglow complexes with multiple coordinated solvents, trans‐complex 1 with both delayed fluorescence (DF) and room temperature phosphorescence (RTP), and cis‐complex 2 with RTP. Remarkably, they can serve as selective and recovery LLPs for l‐Arginine detection, with limit of detection down to 1.0 × 10−7 M. In addition, heating/fumigation can induce reversible arousal/silence of their afterglow, while H2O/DMSO vapor fumigation causes reversible crystalline‐to‐crystalline transformation between them. Detailed mechanism studies reveal that the change in coordinated solvent, including loss/acquisition, exchange, or replacement, plays a key role in such afterglow multi‐stimuli‐responsive properties. This work not only shows the potential of such long‐lived luminescence complex for recovery detection, but also reveals the unique advantages of solvation complex in the preparation of afterglow multi‐stimuli‐responsive materials

A novel tricyanofuran-based near-infrared fluorescent probe for rapid detection and discrimination of Cys/Hcy and GSH/H2S

A novel dual-emission responsive near-infrared fluorescent probe (2-OH-TCF-NBD) for the detection and discrimination of Cys/Hcy, GSH/H2S was developed on the basis of the conjugation of a tricyanofuran-based fluorophore (2-OH-TCF) and 4-chloro-7-nitrobenzofurazan (NBD-Cl). When the probe was added to the Cys/Hcy solution, it resulted in significant fluorescence enhancement at both 527 and 630 nm, while GSH/H2S induced major fluorescence emission only at 630 nm. The detection limits for Cys, Hcy, GSH, and H2S were determined to be 0.029, 0.020, 0.29, and 0.20 μM, respectively. Furthermore, the probe demonstrated some merits including easy preparation, rapid response time, and good anti-interference ability. The possible response mechanism was also proposed and validated by HPLC analysis of the reaction process. Therefore, the probe can be a potential tool for discriminating the detection of Cys/Hcy and GSH/H2S under physiological conditions.

A General Cyanine‐Based Platform for Designing Robust Dual‐Channel Near‐Infrared Fluorescent and Photoacoustic Probes

AbstractThe creation of versatile platforms for developing dual‐channel near‐infrared fluorescent (NIRF) and photoacoustic (PA) probes, especially those engineered to minimize channel crosstalk, is crucial for precise biomarker detection. However, such platforms remain scarce. To bridge this gap, this study introduces an innovative cyanine‐based platform, CySN. The CySN platform showcases remarkable wavelength‐shifting properties, including large fluorescent modality shift (68 nm) and PA modality shift (145 nm) after the decaging reaction. These substantial changes lead to an exceptionally high ratiometric NIRF change of 603‐fold and ratiometric PA change of 261‐fold. Leveraging the CySN platform, dual‐channel NIRF/PA probes have been successfully developed for detecting both small molecule biomarker (H2O2) and enzyme biomarker (esterase). These probes demonstrate the ability to detect their targets through dual‐channel NIRF/PA detection with high sensitivity and selectivity in vitro. Furthermore, the probes effectively harness NIRF signals to image target analytes in living cells. Notably, the probes demonstrate the capability to accurately diagnose tumors by detecting tumor markers (H2O2 and esterase), revealing a 3.6 to 7‐fold ratiometric PA enhancement over normal tissue. Therefore, the CySN platform holds the potential to further advance the development of dual‐channel NIRF/PA probes for biomolecule detection in disease diagnosis.

A near-infrared fluorescent probe for simultaneous detection of pH and viscosity

In this work, we developed a ratiometric fluorescent probe (NT) based on ICT framework in near-infrared (NIR) which could detect pH and viscosity simultaneously. Long emission wavelength in NIR could protect the probe from interference of background fluorescence and improve the accuracy of the test. Due to the presence of thiazole-salt, the probe possessed good water solubility and could respond immediately to pH in water system. The pH values measured by NT in the actual samples were not much different from that measured by the pH meter, therefore, NT could give excellent accuracy. NT realized the reversible detection of pH by protonation and deprotonation. NT was used successfully to detect the pH of actual water samples, human serum and meat, as well as the viscosity variation caused by thickeners. Additionally, NT could monitor the changes of pH and viscosity in living cells. Therefore, the novel probe exhibited potential application in the fields of the environment, human health and food safety evaluation.

Ultrasound-assisted synthesis of a Eu3+-functionalized ZnII coordination polymer as a fluorescent dual detection probe for highly sensitive recognition of HgII and L-Cys

A new ZnII coordination polymer (CP) based on 2,3-pyrazine dicarboxylic acid (H2pzdc) and 4,4′-bipyridine (bpy) (ZCP) was synthesized using a facile slow evaporation method. Single-crystal X-ray diffraction revealed that ZCP is a two-dimensional porous CP, [Zn2(pzdc)2(bpy)(H2O)2] n , with van der Waals forces as the dominant interaction within its layers forming a 63 network. Employing energetic ultrasound irradiation, nanoscale ZCP (nZCP) was successfully synthesized and Eu3+ ions were incorporated within its host lattice (Eu@nZCP). The resulting platform exhibits superior fluorescence characteristics and demonstrates notable optical durability. Therefore, it was used as a dual detection fluorescent sensing platform for the detection of mercury and L-cysteine (L-Cys) in aqueous media through a turn-off/on strategy. In the turn-off process, the fluorescence emission of Eu@nZCP progressively quenches by the addition of HgII via a photo-induced electron transfer (PET) mechanism. The fluorescence of Eu@nZCP is quenched to establish a low fluorescence background through the incorporation of HgII. This devised turn-on fluorescent system is suitable for the recognition of L-Cys (based on the strong affinity of L-Cys to the HgII ion) through a quencher detachment mechanism. This method attained a relatively wide linear range, spanning from 0.001 to 25 µM, with the low detection limit of 5 nM for the sensing of HgII. Also, the corresponding limit of detection (LOD) for L-Cys is 8 nM in a relatively wide linear range, spanning from 0.001 to 40 µM.

Pulsed laser fragmentation synthesis of carbon quantum dots (CQDs) as fluorescent probes in non-enzymatic glucose detection

Pulsed laser fragmentation in liquid (PLFL) is one of the synthesis routes for producing high-purity carbon quantum dots (CQDs) with less toxic by-products in a straightforward system. The limited literature regarding the application of PLFL-synthesized CQDs motivated this study to exploit them as fluorescent probes in glucose sensing. The design of a fluorescent-based non-enzymatic glucose sensor was achieved by implementing CQDs and 3-aminophenylboronic acid (APBA) as fluorescent probes and glucose receptors, respectively. The CQDs were fabricated by PLFL through an Nd:YAG nanosecond laser, starting from the carbon powder dispersion in ethanol. Then, a laser-assisted post-modification process with oxidizing acid was implemented to produce water-dispersible CQDs for easy functionalization. Functionalizing these CQDs with APBA (CQDs-APBA) resulted in a blue-shifted fluorescence emission with a 35% quantum yield and high photostability. The CQDs-APBA exhibited a turn-off response at increasing glucose concentrations. This method offers good sensitivity with a linear detection range of glucose with a concentration of 0.165 to 8 mM and a detection limit of 165 µM. The applicability of this sensor to real analytes such as saliva obtained a satisfactory result with good reproducibility. This work proves that CQDs synthesized from PLFL can also be an alternative fluorescent probe for fluorescent-based sensing applications.

D-π-A Carbzazole Based Reactive Cyano-Substituted C = C bond Probe for Selective and Sensitive Detection of Hydrazine in Aqueous Media.

This work established a newly designed and synthesized carbazole N-phenyl π-conjugated vinyl malononitrile (CPM) fluorescent sensor, which showed typical and remarkable redshift emission properties with different polarity index solvents. Investigative probe CPM is colorimetric and fluorimetric ultrafast and ultrasensitive detection of hazardous hydrazine in an aqueous medium. Furthermore, CPM showed colorimetric and fluorometric responses to interference tests with other amines and high selectivity for detecting hydrazine without interference with other amines in colorimetric and fluorimetric methods. This probe CPM for hydrazine was as low as the lower detection limit value of 2.21 × 10- 8 M. The probe CPM expects significant attention due to its simplicity and cost-effectiveness in detecting hazardous hydrazine. UV-vis, PL, NMR, and MS spectra confirmed the mechanism of probe CPM detection of hazardous hydrazine. However, making a piece test kit attractive for practical hydrazine vapor leak-detection applications is easy. This study can be applied to many pipeline gas transmission industries and transportation facility sectors.

Rapid Identification of Brucella Genus and Species In Silico and On-Site Using Novel Probes with CRISPR/Cas12a

Human brucellosis caused by Brucella is a widespread zoonosis that is prevalent in many countries globally. The high homology between members of the Brucella genus and Ochrobactrum spp. often complicates the determination of disease etiology in patients. The efficient and reliable identification and distinction of Brucella are of primary interest for both medical surveillance and outbreak purposes. A large amount of genomic data for the Brucella genus was analyzed to uncover novel probes containing single-nucleotide polymorphisms (SNPs). GAMOSCE v1.0 software was developed based on the above novel eProbes. In conjunction with clinical requirements, an RPA-Cas12a detection method was developed for the on-site determination of B. abortus and B. melitensis by fluorescence and lateral flow dipsticks (LFDs). We demonstrated the potential of these probes for rapid and accurate detection of the Brucella genus and five significant Brucella species in silico using GAMOSCE. GAMOSCE was validated on different Brucella datasets and correctly identified all Brucella strains, demonstrating a strong discrimination ability. The RPA-Cas12a detection method showed good performance in detection in clinical blood samples and veterinary isolates. We provide both in silico and on-site methods that are convenient and reliable for use in local hospitals and public health programs for the detection of brucellosis.

Engineering a far-red fluorescent probe for rapid detection of Hg(II) ions in both cells and zebrafish

Abnormal accumulation of mercury ions (Hg2+) in organisms can lead to severe central nervous system and other diseases. Therefore, the monitoring and detection of Hg2+ are of great significance for human health and environmental safety. Herein, we designed and synthesized a novel far-red to NIR emission fluorescent probe (Rho-Hg) based on rhodamine derivative as the fluorophore and thiospirolactone as the recognition site for turn-on detecting of Hg2+ in living cells and zebrafish. The probe Rho-Hg displayed superior sensitivity (detection limit = 17.5 nM), rapid response (<1 min), colorimetric change, high selectivity, and moderate pH stability. Leveraging this probe, we realized the real-time monitoring of Hg2+ in real samples, living cells and zebrafish. By fostering zebrafish embryos and larvae in Hg2+-containing nutrient solution, we noticed that Hg2+ was ingested into the zebrafish liver when zebrafish were grown up to 3 days old, and thus we successfully monitored the accumulation and changes of Hg2+ during zebrafish growth and development. Thus, the probe Rho-Hg could be a powerful tool for sensitive and real-time monitoring of Hg2+ in living systems.

An Indocyanine Green-Based Nanoprobe for In Vivo Detection of Cellular Senescence.

There is an urgent need to improve conventional cancer-treatments by preventing detrimental side effects, cancer recurrence and metastases. Recent studies have shown that presence of senescent cells in tissues treated with chemo- or radiotherapy can be used to predict the effectiveness of cancer treatment. However, although the accumulation of senescent cells is one of the hallmarks of cancer, surprisingly little progress has been made in development of strategies for their detection in vivo. To address a lack of detection tools, we developed a biocompatible, injectable organic nanoprobe (NanoJagg), which is selectively taken up by senescent cells and accumulates in the lysosomes. The NanoJagg probe is obtained by self-assembly of indocyanine green (ICG) dimers using a scalable manufacturing process and characterized by a unique spectral signature suitable for both photoacoustic tomography (PAT) and fluorescence imaging. In vitro, ex vivo and in vivo studies all indicate that NanoJaggs are a clinically translatable probe for detection of senescence and their PAT signal makes them suitable for longitudinal monitoring of the senescence burden in solid tumors after chemotherapy or radiotherapy.

An Indocyanine Green‐Based Nanoprobe for In Vivo Detection of Cellular Senescence

AbstractThere is an urgent need to improve conventional cancer‐treatments by preventing detrimental side effects, cancer recurrence and metastases. Recent studies have shown that presence of senescent cells in tissues treated with chemo‐ or radiotherapy can be used to predict the effectiveness of cancer treatment. However, although the accumulation of senescent cells is one of the hallmarks of cancer, surprisingly little progress has been made in development of strategies for their detection in vivo. To address a lack of detection tools, we developed a biocompatible, injectable organic nanoprobe (NanoJagg), which is selectively taken up by senescent cells and accumulates in the lysosomes. The NanoJagg probe is obtained by self‐assembly of indocyanine green (ICG) dimers using a scalable manufacturing process and characterized by a unique spectral signature suitable for both photoacoustic tomography (PAT) and fluorescence imaging. In vitro, ex vivo and in vivo studies all indicate that NanoJaggs are a clinically translatable probe for detection of senescence and their PAT signal makes them suitable for longitudinal monitoring of the senescence burden in solid tumors after chemotherapy or radiotherapy.

Real-time monitoring abscisic acid release from single rice protoplast by amperometry at microelectrodes modified with abscisic acid receptor PYL2

It was previously reported that stress induces a cellular production of abscisic acid in plants, but no direct method shows the evidence. Here, an electrochemical microsensor involving an abscisic acid receptor PYL2 modified carbon fiber microelectrode was fabricated by self-assembly method, where the Cu2+ combined with the histidine tag of PYL2 on the surface of microelectrode was used as the detection probe, the mediated reaction between Cu+ and ferricyanide realized the amplification responses and provided the microsensor with a high sensitivity for detection of abscisic acid with a detection limit of 0.8 nM. With use of this microsensor, an increase of extracellular abscisic acid from single rice protoplast induced by sulfate, osmotic and salinity stress was real-time monitored. Direct measurement of free extracellular abscisic acid in single plant cells might offer important new insights into its role in plants challenged by abiotic stresses.

A lysosome-targeted dual-responsive fluorescent probe for simultaneous detection of NO and H2S in living cells, wild-type C. elegans and zebrafish

A lysosome-targeted dual-responsive fluorescent probe YX-N3-OPD was synthesized to detect two major signaling molecules NO and H2S based on nucleophilic aromatic substitution reaction and reduction simultaneously. The photophysical properties revealed that YX-N3-OPD exhibited strong green fluorescence emission at 530 nm in NO and/or H2S. The specific detection and recognition mechanism of NO and/or H2S was verified by high-resolution mass spectrometry. The probe showed significant fluorescence enhancement in cancer cells, especially in glioblastoma cells (9L/lacZ), mainly accumulated in lysosomes. In addition, YX-N3-OPD was also applied for NO and H2S monitoring in wild-type C. elegans and zebrafish with relatively high selectivity and sensitivity.