Sensitive Molecular Probes Research Articles

Evaluation of 99m Tc-MccJ25 peptide analog in mice bearing B16F10 melanoma tumor as a diagnostic radiotracer.

Objective(s):Despite recent advances in treatment modalities, cancer remains a major source of morbidity and mortality throughout the world. Currently, the development of sensitive and specific molecular imaging probes for early diagnosis of cancer is still a problematic challenge. Previous studies have been shown that some of the antimicrobial peptides (AMPs) exhibit a broad spectrum of cytotoxic activity against cancerous cells in addition to their antimicrobial activities. MicrocinJ25 (MccJ25) is an antimicrobial peptide that is produced by Escherichia coli (E. coli) strain. The aim of this study was to investigate the potential of a new peptide radiopharmaceutical derived from MccJ25 for diagnosis of melanoma tumor bearing C57BL/6 mice.Methods:A 14 amino acid analog of MccJ25 was labeled with technetium-99m (99mTc) through hydrazinonicotinamide (HYNIC) chelator and tricine as coligand. In vivo tumor uptake and tissue distribution were evaluated. The in vivo biodistribution studies were determined in C57BL/6 mice bearing B16F10 tumor.Results:The amount of non-peptide related 99mTc-impurities that measured by thin layer chromatography (TLC) did not exceed 5% of the total radioactivity. The in vitro binding to B16F10 cells was 30.73 ± 0.9% after 1 h incubation at 37°C, and saturation binding experiments showed good affinity for radio-complex (Kd=47.98±6.25 nM). The melanoma tumor was clearly visible up 1 h post-injection by gamma camera imaging.Conclusion:The results showed that 99mTc-labeld peptide could be a promising candidate as a targeting radiopharmaceutical for melanoma tumor imaging in mice.

Fluorescence lifetime imaging of upper gastrointestinal pH in vivo with a lanthanide based near-infrared τ probe.

Time-resolved fluorescence lifetime imaging (FLIM) in the near-infrared region of 900-1700 nm not only allows a deep tissue penetration depth but also offers the unique benefit of the quantitative visualization of molecular events in vivo and is independent of local luminescence intensity and fluorophore concentration. Herein, we report the design of a wide-range pH sensitive molecular probe based on Yb3+ porphyrinate. The Yb3+ probe shows increasing NIR emission and lifetime with pK a values of ca. 6.6 from pH 9.0 and 5.0 and also displays an elongated lifetime from ca. 135 to 170 μs at lower pH values (5.0-1.0) due to aggregation and reduced exposure to water at low pH values. Importantly, the probe is able to monitor a wide range of in vivo gastrointestinal pH values in mice models and the potential applications in imaging-guided gastrointestinal diagnostics and therapeutics were revealed. This study shows that lifetime contrast is important for preclinical imaging; lanthanide complexes could be successfully used in the design of stimuli-responsive NIR τ probes for advanced in vivo imaging.

A Substitution‐Dependent Light‐Up Fluorescence Probe for Selectively Detecting Fe3+ Ions and Its Cell Imaging Application

AbstractDeliberate design of specific and sensitive molecular probes with distinctive physical/chemical properties for analyte sensing is of great significance. Herein, by taking advantage of the position‐dependent substituent effects, an aggregation‐induced emission featured iron (III) probe from ortho‐substituted pyridinyl‐functionalized tetraphenylethylene (TPE‐o‐Py) is synthesized. It displays high sensitivity and selectivity toward iron (III) detection. The recognition arises from the position isomer of ortho‐substitution, and the fact that TPE‐o‐Py has a low acid dissociation constant (pK a) that is close to that of hydrolyzed Fe3+. Importantly, TPE‐o‐Py as a light‐up fluorescence probe could be employed for Fe3+ sensing in living cells with a pronounced red‐shift in fluorescence emission.

Calix[4]arene Based Redox Sensitive Molecular Probe for SERS Guided Recognition of Labile Iron Pool in Tumor Cells.

Targeting the intracellular "labile" iron pool is turned as a key modulator for cancer progression since the former is responsible for several pathological processes in tumor cells. Herein, we report a nonfluorescent calix[4]arene based triazole appended molecular probe (PTBC) for redox-specific detection of Fe3+ under physiological condition by UV-vis, FT-IR, 1H NMR, HR-MS spectroscopies, ITC, and the binding strategy between Calix[4]arene and Fe3+ was modeled by DFT calculations. As a new insight PTBC probe showed significant Raman fingerprint through surface enhanced Raman scattering (SERS) modality revealing the ultrasensitive detection of Fe3+ with a LOD of 2 nM. Interestingly, intracellular "iron pool" has been recognized in human lung adenocarcinoma cells (A549) by the PTBC illustrating the distinct Raman mapping. Finally, PTBC imparted cytotoxicity via reactive oxygen species (ROS) generation in cellular milieu signifies its capability as a theranostic molecular probe.

Stern-Layer Adsorption of Oligonucleotides on Lamellar Cationic Lipid Bilayer Investigated by Polarization-Resolved SFG-VS.

The molecular interaction between the oligonucleotides and lipid membranes is the key to the functions of virus, aptamer, and various oligonucleotide-based materials. In this study, the conformational changes of oligonucleotides (dT25) on lamellar cationic 1,2-dimyristoyl-3-trimethylammonium-propane (DMTAP) bilayer were investigated by polarization-resolved sum frequency generation vibrational spectroscopy (SFG-VS) in situ. The SFG-VS spectra within different wavenumber ranges were analyzed to give conformation details of thymine groups, phosphate groups, and OD/OH groups and to provide a comprehensive and fundamental understanding of the oligonucleotide adsorption on a model bilayer. It is shown that the adsorption of dT25 on DMTAP bilayer reaches maximum at CdT ≈ 500 nM. And the conformation of dT25 molecules change significantly when surface charge of DMTAP bilayer reaches the point of zero charge (PZC) at CdT ≈ 100 nM. Combined spectroscopic evidences also indicate that the formation of electric double layer at the DMTAP/dT25 surface follows the Gouy–Chapman–Stern model. The analysis results also show that the symmetric PO2– stretching mode of oligonucleotide molecules can serve as a sensitive vibration molecular probe for quantifying the oligonucleotide/lipid charge ratio and determine the point of zero charge (PZC) of lipid bilayer surface, which may help researchers to control the layer-by-layer assembly of oligonucleotide–lipid complexes and to improve the efficiency genetic therapy against cancer and viral infections.

Minimal Residual Disease Assessment and Risk-based Therapy in Acute Lymphoblastic Leukemia.

The study of minimal residual disease (MRD) in adult patients with acute lymphoblastic leukemia (ALL) allows a greater refinement of the individual risk classification and is the best support for risk-specific therapy with or without allogeneic hematopoietic cell transplantation (HCT). Using case-specific sensitive molecular probes or multiparametric flow cytometry on marrow samples obtained from the end of induction until midconsolidation, MRD assays can detect up to 1 leukemic cell of 10,000 total mononuclear cells (sensitivity, 0.01%; ie,≥104). This cutoff, presently bound to technical limitations and subject to improvement, reflects the individual chemosensitivity and is strongly correlated with treatment outcome. The chance for cure is approximately 70% in the MRD-negative subset but only 20% to 30% in MRD-positive patients, in any diagnostic and risk subset. As shown by prospective trials from Germany, Italy, Spain, and France-Switzerland-Belgium, approximately 50% to 70% of unselected adult patients with Philadelphia-negative ALL achieve and maintain an early MRD response, whereas the remainder do not, including a substantial proportion of clinically standard-risk patients, and require an HCT to avert at least partially the risk of relapse. Along with the diffusion of more effective "pediatric-inspired" chemotherapy programs, the MRD analysis is an integral part of a modern management strategy, guiding the decision process to transplant or not, in which case nonrelapse mortality using HCT in first remission-still 10% to 20%-is totally abolished. The use of new agents such as monoclonal antibodies, small inhibitors, and chimeric antigen receptor T cells is opening a new era of MRD-directed therapies, that will further increase survival rates.

Environmentally sensitive molecular probes reveal mutations and epigenetic 5-methyl cytosine in human oncogenes.

There is currently an unmet need for reliable tools that allow for direct detection and quantification of modifications in genomic DNA. For example, in cancer research and clinical diagnostics, target DNA has to be amplified and sequenced in order to reveal mutations. For 5-methylcytosine detection, bisulfite treatment of DNA is applied for the analysis, which often leads to poor specificity and reproducibility of the results. Herein we describe a simple approach that specifically detects clinically significant modifications in the human oncogenes BRAF and KRAS. We prove that this can be done using a fast and reliable hybridization assay applying novel internally labelled oligonucleotide probes and optical detection methods.

NMR Hyperpolarization Techniques of Gases.

Nuclear spin polarization can be significantly increased through the process of hyperpolarization, leading to an increase in the sensitivity of nuclear magnetic resonance (NMR) experiments by 4-8 orders of magnitude. Hyperpolarized gases, unlike liquids and solids, can often be readily separated and purified from the compounds used to mediate the hyperpolarization processes. These pure hyperpolarized gases enabled many novel MRI applications including the visualization of void spaces, imaging of lung function, and remote detection. Additionally, hyperpolarized gases can be dissolved in liquids and can be used as sensitive molecular probes and reporters. This Minireview covers the fundamentals of the preparation of hyperpolarized gases and focuses on selected applications of interest to biomedicine and materials science.

MMP2-sensing up-conversion nanoparticle for fluorescence biosensing in head and neck cancer cells

Upconversion nanoparticles (UCNPs) have extensive biological-applications because of their bio-compatibility, tunable optical properties and their ability to be excited by infrared radiation. Matrix metalloproteinases (MMPs) play important roles in extracellular matrix remodelling; they are usually found to significantly increase during cancer progression, and these increases may lead to poor patient survival. In this study, we produced a biosensor that can be recognized by MMP2 and then be unravelled by the attached quencher to emit visible light. We used 3.5-nm gold nanoparticles as a quencher that absorbed emission from UCNPs at a wavelength of 540nm. The biosensor consists of an upconversion nanoparticle, MMP2-recognized polypeptides and quenchers. Here, UCNPs consisting of NaYF4:Yb3+/Er3+ were prepared via a high temperature co-precipitation method while protecting the oleic acid ligand. To improve the biocompatibility and modify the UCNPs with a polypeptide, they were coated with a silica shell and further conjugated with MMP-recognizing polypeptides. The polypeptide has two ends of featuring carboxylic and thiol groups that react with UCNPs and AuNPs, and the resulting nanoparticles were referred to as UCNP@p-Au. According to the in vitro cell viability analysis, UCNP@p-Au exhibited little toxicity and biocompatibility in head and neck cancer cells. Cellular uptake studies showed that the MMP-based biosensor was activated by 980-nm irradiation to emit green light. This MMP-based biosensor may serve as sensitive and specific molecular fluorescent probe in biological-applications.

Cysteine-Mediated Intracellular Building of Luciferin to Enhance Probe Retention and Fluorescence Turn-On.

The development of sensitive and selective small molecular probes that enable real-time detection of endogenous cysteine (Cys) has become an attractive topic because of the essential roles played by Cys in controlling the cellular nitrogen balance and in maintaining biological redox homeostasis. Herein, we report a Cys-specific probe, 2-cyanobenzothiazol-6-yl acrylate (CBTOA), that shows not only fluorescence turn-on for sensitive detection of endogenous Cys but also enhanced probe retention inside cells for real-time monitoring of Cys levels upon external stimulation. Cys-mediated intracellular formation of luciferin from CBTOA was the key strategy leading to this new type of fluorogenic probe. CBTOA showed fast response to Cys in living cells and liver tissue slices with high sensitivity and selectivity. By using CBTOA as a real-time probe, we were able to monitor the change in Cys levels in living HeLa cells under ROS-induced oxidative stress as well as in human mesenchymal stem cells during adipogenic differentiation.

Design, Synthesis, and Evaluation of Highly Sensitive Molecular Probes for Magnetic Resonance Analysis

Design, Synthesis, and Evaluation of Highly Sensitive Molecular Probes for Magnetic Resonance Analysis

Metal-clad optical waveguide fluorescence device for the detection of heavy metal ions

We developed Hg 2+ -sensing chips by decorating the external surface of metal-clad optical waveguides with a monolayer of Hg 2+ -sensitive fluorescent molecular probes. The emission properties of the original water-soluble form of the molecule were previously found to be selectively quenched in the presence of Hg 2+ ions. The fabricated samples were tested with optical waveguide fluorescence spectroscopy by putting them in contact with a 5-μM water solution of Hg 2+ ions and recording the emission spectra versus incubation time. The estimate of the limit of detection was 150 nM. A preliminary evaluation of the selectivity of the structure was also performed by using Cd 2+ as possible interfering analytes.

1α,25-二羟基维生素D<sub>3</sub>及其类似物的 合成研究进展

许多合成的维生素D类似物正在被应用当作新的治疗药物和灵敏的生物分子探针，本文综述了活性维生素D3即1α,25-二羟基维生素D3及其类似物的合成方法研究进展。 An increasing number of synthetic Vitamin D analogues are now being used as new powerful drugs for treatment of various human diseases and also as sensitive molecular biology probes. This paper reviews the synthetic methods and advances in synthesis of 1α,25-dihydroxyvitamin D3 and related analogues.

Spectroscopic probing of the microenvironment of 7-oxy(5-selenocyanato-pentyl)-2H-1-benzopyran-2-one in ionic and nonionic micelles

• Modulation of photophysics in different micellar assembly. • Rotational behavior in confined media. • Dramatic enhancement of the PCM fluorescence in the CTAB micelles upon addition of quencher. • Ion exchange phenomenon operative at the micelle surface. • Development of highly polarity sensitive fluorescence molecular probe. Entrapping of a drug molecule having prospective therapeutic activity in microhetrogeneous environments and modulating important photophysical properties is of immense importance in various potential applications. The photophysical properties of a newly synthesized bioactive 7-oxy(5-selenocyanato-pentyl)-2H-1-benzopyran-2-one (PCM) has been studied in micellar solutions of anionic sodium dodecyl sulfate (SDS), cationic cetyl trimethyl ammonium bromide (CTAB) and nonionic p-tert-octylphenoxy polyoxyethanol (TX-100) micelle using steady state, time resolved fluorescence and fluorescence anisotropy techniques. The present effort illustrates the degree of accessibility of the fluorophore toward the heavy ion quencher in the presence of micelles of different surface charge characteristics. Iodide induced quenching leads to a decrease in the fluorescence intensity in case of SDS and TX-100 micelles, where as in CTAB micelles enhancement of fluorescence intensity is observed. Quantitative estimation of the micropolarity at the binding site of the probe has been determined. The imposed motional restriction of the microenvironment around the probe has been established from environment dependent fluorescence parameter, fluorescence anisotropy ( r ) value.

Mechanical Control of Renilla Luciferase

We report experiments where the activity of the enzyme luciferase from Renilla reniformis is controlled through a DNA spring attached to the enzyme. In the wake of previous work on kinases, these results establish that mechanical stress applied through the DNA springs is indeed a general method for the artificial control of enzymes, and for the quantitative study of mechano-chemical coupling in these molecules. We also show proof of concept of the luciferase construct as a sensitive molecular probe, detecting a specific DNA target sequence in an easy, one-step, homogeneous assay, as well as SNP detection without melting curve analysis.

Synthesis and spectroscopic properties of 4-amino-1,8-naphthalimide derivatives involving the carboxylic group: a new molecular probe for ZnO nanoparticles with unusual fluorescence features

Of a series of 4-substituted 1,8-naphthalimides, fluorescent 4-(6-piperidinyl-1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)benzoic acid (4) was found to be a sensitive molecular probe for ZnO nanoparticles. We investigated in detail one- and two-photon absorption properties of this fluorophore. In nonpolar solvents, the acid 4 absorbs at about 400 nm and fluoresces at 500 nm with a fluorescence lifetime of about 7 ns, similar to the ester 6 and typical of the lifetimes of other derivatives of this type. Although the anionic form of this acid is not fluorescent, partial ionization of 4 in polar solvents, such as ethanol and acetonitrile, is not only accompanied by the expected decrease in the fluorescence quantum yield, but also gives rise to bathochromic shifts of both absorption and fluorescence and dual fluorescence with lifetimes of 0.2–0.3 ns and 6 ns ascribed to the formation of anionic complexes. The interaction with the ZnO surface brings about further considerable changes in the fluorescence patterns.

Micro-CT Molecular Imaging of Tumor Angiogenesis Using a Magnetite Nano-Cluster Probe

Due to its high resolution, micro-CT is desirable for molecular imaging of tumor angiogenesis. However, the sensitivity of micro-CT to contrast agents is relatively low. Therefore, the purpose of this study is to develop high micro-CT sensitive molecular imaging probes for direct visualization and dynamic monitoring of tumor angiogenesis. To this end, Arg-Gly-Asp (RGD) peptides conjugated magnetite nano clusters (RGD-MNCs) were developed by assembling individual magnetite nano particles into clusters with amphiphilic (maleimide) methoxypoly(ethylene glycol)-b-poly(lactic acid) ((Mal)mPEG-PLA) copolymer and subsequently encoding RGD peptides onto the clusters for specific targeting alpha(v)beta3 integrin. The hydrodynamic size of RGD-MNCs was about 85 nm. To test its specificity, alpha(v)beta3 positive cells (H1299) were incubated with magnetite nano clusters (MNCs), RGD-MNCs or RGD-MNCs competition with free RGD peptides. Prussian Blue staining and inductively coupled plasma optical emission spectrometer (ICP-OES) measurements indicated that the cell uptake of RGD-MNCs was significantly more than that of MNCs, which could be inhibited by free RGD peptides. For detection of tumor angiogenesis, mice bearing H1299 tumors were injected intravenously with RGD-MNCs at the dose of 400 micro mol Fe/kg. Tumor angiogenic hot spots as well as individual angiogenic vessels could be clearly manifested by micro-CT imaging 12 h post injection, which was dynamically monitored with the extension of probe circulation time. Subsequent histological studies of tumor tissues verified that RGD-MNCs registered tumor angiogenic vessels. Our study demonstrated that RGD-MNC probes fabricated in this study could be used to effectively target alpha(v)beta3 integrin. Using high resolution micro-CT in combination with the probes, tumor angiogenesis could be studied dynamically.

Simple and highly sensitive molecular beacon probe based on target-induced structure-switching DNA for mercury(ii) detection

A simple and highly sensitive molecular beacon (MB) probe, based on coupling target-induced conformational switching of DNA hairpins with thymine–Hg2+–thymine (T–Hg2+–T) coordination chemistry, was designed for the determination of mercury(II) (Hg2+). In this MB, the DNA hairpins contain a stem of 6 base pairs enclosing a 27 nucleotide (nt) loop and an additional 12 nt sticky end at the 3′ end. The twelfth base from the 3′ end was labeled with a quencher of 4-(4′-dimethylaminophenylazo) benzoic acid (DABCYL), and the 5′ end was labeled with a fluorophore fluorescein amidite (FAM). In the absence of Hg2+, the DNA hairpin is in a closed form resulting in fluorescence quenching. In the presence of the target analyte, Hg2+-mediated base pairs induce the conformational change from the sticky end to open the hairpins; the fluorophore and the quencher are moved away from each other, leading to the restoration of fluorescence. The dynamic concentration range was from 0.5 to 50 nM Hg2+ with a detection limit of 0.3 nM at the 3σ level. The assay strategy afforded high selectivity for Hg2+ against other environmentally related metal ions. The developed method is very simply, only requiring a one-step reaction to realize the assay of Hg2+.

π-Conjugated polymer–Eu3+complexes: versatile luminescent molecular probes for temperature sensing

We report π-conjugated polymer–Eu3+ ion complexes as new potential luminescent thermo-sensitive molecular probes. Carboxylic acid functionalized segmented π-conjugated polymers having oligophenylenevinylene (OPV) chromophores in the poly(ethyleneoxide) or polymethylene backbones were custom designed, synthesized and utilized as efficient photosensitizers for Eu3+ ions. These π-conjugated polymer–Eu3+ ion complexes were found to be thermo-sensitive and behaved as reversible ‘turn-on’ or ‘turn-off’ luminescent switches in solution and in solid state. Luminescent decay studies revealed that the red-emission from the Eu3+ ion excited state was highly sensitive to temperature which drove the functioning of optical switches. The decay rate constants followed a typical Arrhenius trend over a wide temperature range having similar activation energies. Both the nature as well as length of the segmented polymer chain that tied the OPV optical chromophores in the backbone determine the temperature range of the luminescent on–off process. The emission characteristics of the oligomer–Eu3+ ion complex were found to be non-thermosensitive which emphasized the need for the segmented π-conjugated polymer ligand structure for the probes based on Eu3+ ion complexes. The present strategy opens up new concept and molecular design principles for π-conjugated polymer–lanthanide ion complexes as potential candidates for temperature sensitive luminescent molecular probes.

Assessing Breast Cancer Margins Ex Vivo Using Aqueous Quantum-Dot-Molecular Probes

Positive margins have been a critical issue that hinders the success of breast- conserving surgery. The incidence of positive margins is estimated to range from 20% to as high as 60%. Currently, there is no effective intraoperative method for margin assessment. It would be desirable if there is a rapid and reliable breast cancer margin assessment tool in the operating room so that further surgery can be continued if necessary to reduce re-excision rate. In this study, we seek to develop a sensitive and specific molecular probe to help surgeons assess if the surgical margin is clean. The molecular probe consists of the unique aqueous quantum dots developed in our laboratory conjugated with antibodies specific to breast cancer markers such as Tn-antigen. Excised tumors from tumor-bearing nude mice were used to demonstrate the method. AQD-Tn mAb probe proved to be sensitive and specific to identify cancer area quantitatively without being affected by the heterogeneity of the tissue. The integrity of the surgical specimen was not affected by the AQD treatment. Furthermore, AQD-Tn mAb method could determine margin status within 30 minutes of tumor excision, indicating its potential as an accurate intraoperative margin assessment method.