Small Probe Research Articles

Benzo[c,d]Indole-Quinoline-Based Deep-Red Emissive Probes for Live-Cell Imaging of Nucleolar RNA.

Small molecular weight fluorescent probes capable of binding to RNAs have been powerful tools for understanding the intracellular behaviors of RNAs. In this chapter, we describe the fluorescence imaging of nucleolar RNA in living cells using deep-red emissive probes with benzo[c,d]indole-quinoline (BIQ) monomethine cyanine scaffolds. These probes feature a significant fluorescence "off-on" ability upon binding to RNAs (>100-fold) in the deep-red spectral region (λem>650nm). In addition, they have many advantages for fluorescence sensing of RNAs and nucleolar RNA imaging, such as longer emission wavelength, higher photostability, and better counterstaining compatibility for live cell imaging, compared to a commercially available RNA-binding probe, SYTO RNA select.

A biocompatible fluorescent probe for endogenous hydrogen sulfide detection and imaging.

Hydrogen sulfide (H2S) acts as a messenger molecule and can mediate a variety of physiological functions. Conventional methods are seldom used to detect endogenous H2S and present some difficulties in selective and accurate detection. Reaction-based recognition of endogenous H2S by organic small molecule probes with good specificity and biocompatibility. To address this challenge, we developed a novel H2S fluorescent probe 4-(2-(6-hydroxy-2-naphthyl) ethyl)-1-methylpyridinium (DSNP) that triggers a thiolysis reaction through a strong electron withdrawing group, releasing a fluorescent molecule. The simple probe DSNP not only have good selectivity, large Stokes shifts and biocompatibility, but also demonstrated a detection limit as low as 28.4 nM and reaction times as quick as 30 minutes. Moreover, it has been successfully applied to imaging intracellular H2S in myeloma cells and zebrafish. This study opens new insights to help push this probe forward for its applicability for detailed H2S localization studies in osteosarcoma.

Direct Visualization of Hepatitis C Virus RNA in Living Cells and Mice.

RNA virus infection is a global health issue with a significant economic burden. Direct visualization of the viral RNA genome in living cells is crucial for virological research and early clinical diagnosis. Thus, the need to continue research to find imaging toolkits is urgent. The RNA G-quadruplex (G4), a noncanonical secondary structure with stacked planar G-quartets, has recently been demonstrated in the RNA genomes of various viruses, especially the hepatitis C virus (HCV). Recent advancements in small molecular fluorescent probes have paved the way for a novel method of visualizing RNA G4s. Herein, we describe a fluorogenic probe-based RNA G4s light-up system for visualizing the HCV genome in living host cells and HCV RNA-presenting mini-organ-bearing mice without complicated sample pretreatment. Using this approach, we achieved (i) the visualization of HCV RNA genome in living cells, (ii) the investigation of viral RNA subcellular distribution, (iii) the dynamic tracking of native HCV infection and propagation in the host cell, and (iv) the high-contrast HCV RNA imaging in living mice bearing the HCV RNA-presenting mini-organ.

Tissue-probe contact assessment during robotic surgery using single-fiber reflectance spectroscopy

The introduction of robotic surgery has improved minimally invasive surgery, and now robotic surgery is used in several areas of surgical oncology. Several optical techniques can be used to discriminate cancer from healthy tissue based on their optical properties. These technologies can also be employed with a small fiber-optic probe during minimally invasive surgery; however, for acquiring reliable measurements, some optical techniques require the fiber-optic probe to be in direct contact with the tissue. The lack of tactile feedback in robotic surgery makes assessing tissue-probe contact suitable for optical contact measurements challenging for the surgeon. In this study, we investigated the use of single fiber reflectance (SFR) to determine tissue-probe contact adequately. A machine learning-based algorithm was developed to classify if direct tissue-probe contact was present during the measurement in an ex-vivo tissue setup. Using this classification algorithm, an average accuracy of 93.9% was achieved for assessing probe-tissue contact, suggesting that this technique can be utilized to assess tissue-probe contact in an in vivo clinical setting.

Application of a large wall electric probe (LWEP) for plasma and ambient gas studies

Abstract The operation of a large wall electric probe (LWEP) is analyzed, where the probe surface area in contact with the plasma is only a few times smaller than the area of the plasma boundaries. Compared to a small standard Langmuir probe (SLP), the LWEP may have significantly greater resolution and sensitivity, but could substantially perturb the plasma and distort the measured properties. A target application for the LWEP is where the sensitivity of the measurement is critical, but the effects of the plasma perturbation is minimal. A specific case where a LWEP may outperform a SLP is the measurement of a nonlocal electron distribution function at energies of electron free diffusion, that is, at energies significantly exceeding thermal electron energies, in order to obtain information about the parameters of the plasma or ambient gas. Examination of the LWEP analysis process has revealed that, depending on the plasma volume and probe configurations, it may be necessary to measure either the first or second derivatives of the probe current with respect to the probe potential to derive the energetic part of the electron energy distribution function.

Research progress of organic small probes sensitive to tumor microenvironment

The development, metastasis, and spread of malignant tumors are closely associated with the inherent properties of tumor cells. These tumors often exhibit low pH, low polarity, high viscosity, and hypoxia. Understanding these characteristics is essential for early cancer detection and monitoring treatment effectiveness. Small molecule fluorescent probes have emerged as powerful tools for real-time localization, dynamic detection, and visualization of tumors. In this paper, we review recent progress in the use of these probes to study tumor biology, focusing on their design strategies, response mechanisms, and preclinical applications for detecting pH, viscosity, polarity, and hypoxia.

Design and Synthesis of Small Molecule Probes of MDA-9/Syntenin.

MDA-9/Syntenin, a key scaffolding protein and a molecular hub involved in a diverse range of cell signaling responses, has proved to be a challenging target for the design and discovery of small molecule probes. In this paper, we report on the design and synthesis of small molecule ligands of this key protein. Genetic algorithm-based computational design and the five-eight step synthesis of three molecules led to ligands with affinities in the range of 1-3 µM, a 20-60-fold improvement over literature reports. The design and synthesis strategies, coupled with the structure-dependent gain or loss in affinity, afford the deduction of principles that should guide the design of advanced probes of MDA-9/Syntenin.

β-galactosidase instructed in situ self-assembly fluorogenic probe for prolonged and accurate imaging of ovarian tumor

Fluorescent probes are essential for optical imaging and have been extensively employed for precise cancer diagnosis studies. β-galactosidase (β-gal) serves as a primary biomarker for ovarian cancer and has been utilized to develop imaging probes for accurate tumor diagnosis. However, traditional small molecular probes have limitations in terms of rapid diffusion and metabolic clearance from the target lesion, resulting in a short imaging window and compromised tumor-to-background ratios (TBR). Herein, we integrated an enzyme-instructed in situ self-assembly strategy to construct Gal-IRFF, a small molecule-based activatable near-infrared (NIR) fluorogenic probe. Upon cleavage by endogenous β-gal overexpressed in ovarian cancer cells, IRFF exhibited enhanced NIR fluorescence signals and self-assembled into nanoparticles through intermolecular interactions of the Phe-Phe (FF) dipeptide moiety, which facilitated probe accumulation and retention within the tumor lesion. Compared with the small molecule probe Gal-IR, our proposed self-assembly probe Gal-IRFF demonstrated a lower limit of detection (LOD) towards β-gal and showed remarkable improvements in distribution and retention time within SKOV3 cells in vitro and tumors in vivo, thereby providing a long-term imaging window for real-time monitoring β-gal levels in ovarian tumors. Therefore, this study highlights the potential of an enzyme-instructed self-assembly fluorogenic probe design approach for achieving precise tumor diagnosis in vivo.

Storehouse of Peculiar Supramolecular Architecture: Probing the Charge-Transfer Phenomenon and Effect of Altering the pH in a Meta-Oriented Single Donor-Double Acceptor Fluorophore.

The investigation of excited-state intramolecular charge transfer (ESICT) has been a fascinating area of research. Although the ESICT events have been studied mostly for para-disubstituted donor-acceptor type molecules, the meta-oriented donor-acceptor type molecules have also shown tremendous potential as ESICT active molecules. In the current work, a small fluorescent probe diethyl 5-amino isophthalate (DE-5A-IPA) was investigated as a potential model to investigate ESICT events in the solution as well as solid phase. DE-5A-IPA was synthesized easily starting from commercially available 5-amino isophthalic acid in excellent yield. In the solid state, differing extents of CH···π-type binding led to formation of fully planar and puckered "cyclobutane" mimic supramolecular architecture, as evidenced from the crystal structure analysis. In addition, the single crystal structure of DE-5A-IPA shows that the donor (-NH2) and acceptor (-CO2Et) are situated in the same plane, thereby assuring the prerequisites of a through-space charge transfer. DE-5A-IPA undergoes noticeable Stokes shift (in cm-1) when the polarity of the medium was changed (4820 cm-1 in hexane; 9375 cm-1 in water). The excited-state dipole moment (μe) was calculated to be 4.0 units higher than the ground-state dipole moment (μg). DE-5A-IPA had an appreciable quantum yield (0.10 ± 0.03 to 0.27 ± 0.04). The ESICT phenomenon was also investigated by ground- and excited-state structural calculations using Gaussian 16 software. The excited-state lifetime measured by the time correlated single photon counting technique was found to vary with the polarity of the solvent, thereby providing further support to the ESICT phenomenon being operative in DE-5A-IPA. Taking advantage of the -NH2 group (which is susceptible to protonation) in DE-5A-IPA, steady state and time-resolved photodynamics were investigated in solutions of varying pH values. Interestingly, the emission quantum yields as well as the emission lifetime increase with an increase in pH value, thereby establishing DE-5A-IPA as a pH-sensitive probe. The current findings shall boost the understanding of meta-oriented ESICT enabled compounds in terms of their excited-state photophysics.

Activity-Based DNA-Encoded Library Screening for Selective Inhibitors of Eukaryotic Translation.

Small molecule probes exist for only ∼2% of human proteins because most lack functional binding pockets or cannot be assayed for high-throughput screening. Selective translation modulation circumvents canonical druggability and assay development constraints by using in vitro transcription-translation (IVTT) as a universal biochemical screening assay. We developed an IVTT activity assay by fusing a GFP reporter to various target gene sequences and screened the target sequences for inhibitors in microfluidic picoliter-scale droplets using a 5,348-member translation inhibitor DNA-encoded library (DEL). Screening a proof-of-concept PCSK9-GFP reporter yielded many hits; 6/7 hits inhibited PCSK9-GFP IVTT (IC50 1-20 μM), and the lead hit reduced PCSK9 levels in HepG2 cells. Preliminary selectivity was informed by counterscreening the DEL against a frameshift mutant PCSK9-GFP reporter. A plug-and-play approach to assay development and screening was demonstrated by scouting the DEL for activity using reporter genes of targets with difficult-to-assay or even unknown function (RPL27, KRASG12D, MST1, USO1). This microfluidic IVTT DEL screening platform could scale probe discovery to the human proteome and perhaps more broadly across the tree of life.

An intramolecular charge transfer based fluorescent probe for imaging of OCl–

The discovery and utilization of new fluorescent chromophore is indispensable to exploit high performance probes for biological research. Stokes shift is one of the most important properties of chromophore accounting for super-resolution fluorescence imaging. Intramolecular charge transfer (ICT) is one of the fundamental mechanisms for fluorescence that accompanied by large Stokes shifts. Based on the conformational changes between ground and excited states, ICT models can be divided into two types: conformation-steady ICT, whose conformation remains unchanged, and conformation-changeable ICT, which is characterized by the rotation of the chromophore around an axis upon excitation. Herein, we report a new chromophore whose donor and acceptor parts took a butterfly geometry with a dihedral angle of 21° in ground state and a planar conformation upon photo excitation. The bent conformation might be ascribed to the extra conjugated double bond, which made the coplanarity of the chromophore in ground state get worse. The chromophore shows a remarkable Stokes shift over 150 nm and a high fluorescence quantum yieldof 0.62. The limit of detection is 41 nM, which enabled the imaging of basal as well as induced OCl– in different cells. Moreover, the pronounced spectroscopic properties ensure the in vivo monitoring of OCl– in arthritic mice. This finding would shed light on the exploitation of small molecule probes based on new fluorescence chromophore for precise biological imaging.

Preliminary Evaluation of the Value of a Small-Molecule Probe Targeting DNMT1 in Detecting the Methylation of PAX1 in Cervical Cancer.

To investigate the screening value of a small-molecule probe to assess the methylation of PAX1 in cervical cancer. The diagnostic threshold of the grayscale values for cervical lesions was assessed by plotting the Receiver Operating Characteristic (ROC) curve of subjects. Grayscale values were significantly different among the four groups (p<0.05). Compared with the LSIL and cervicitis groups, a considerably higher grayscale value was found in the CA and high-grade squamous intraepithelial lesion (HSIL) groups (both p<0.05). The differential ROC curves of the grayscale values showed that the diagnostic Area Under Curve of the probe for cervicitis and low-grade squamous intraepithelial lesion (LSIL) was 0.8724 (95% CI=0.7762-0.9685, p<0.0001), for cervicitis and CA was 1.0000 (p<0.0001), for the LSIL and HSIL was 0.5484 (95% CI = 0.3826-0.7142, p=0.5755), and for the LSIL and CA was 0.7724 (95% CI = 0.6016-0.9432, p = 0.0138). The small molecular probe has certain application value in differentiating the type of cervical lesions and has better efficacy in distinguishing cervical inflammatory and precancerous lesions from carcinogenesis, but less efficacy in determining the type of precancerous lesions.

Red fluorescent AIE bioprobes with a large Stokes shift for droplet-specific imaging and fatty liver diagnosis

Lipid droplets (LDs) as spherical dynamic subcellular organelles, play an important role in various cellular functions such as protein degradation, lipid metabolism, energy storage, signal transduction, and membrane formation. Abnormal function of LDs will lead to a series of diseases and hence monitoring the status of LDs is particularly important. In this study, we synthesized a water-insoluble red fluorescent emitting small molecule fluorescent probe (TPE-TCF), which exhibited aggregation-induced emission (AIE) properties and enabled highly selective real-time imaging of LDs (Pearson’s R value was 0.90). More interestingly, this probe was able to track the dynamic processes of LDs in living cells, including lipophagy, and monitor fatty liver disease in mice. Therefore, TPE-TCF with red fluorescence emission, good biocompatibility, large Stokes shift, AIE properties, LDs imaging, and fatty liver recognition capabilities can be practically used in more LDs-related diseases.

Mechanical Robustness Analysis of Semiconductors with a Single Needle Probe Card Using Acoustic Emissions

The combination of indentation testing and acoustic emission (AE) is widely used to analyze the fracture toughness of test substrates. In the manufacturing of semiconductor devices this material parameter also plays an important role. During the so-called wafer testing inside a wafer prober small probe tips are pressed onto the chip surface to check its performance. To prevent damaging the chip by that, the fracture toughness and load limit of it has to be defined in a prequalification step. This paper presents a test system that imitates the wafer testing process as close as possible and uses acoustic emission to detect appearing cracks and thereby also the load limit. The frontend of the test setup consists of a modular single needle probe card which can be placed in a wafer prober. Also, the indenter properties (tip diameter, stiffness, etc.) can be adjusted to the probe used later on during productive wafer testing to ensure realistic probing conditions. A piezoelectric sensor and a full strain gauge Wheatstone bridge are implemented close to the single indenter to measure the AEs and the applied contact force respectively. The signal-to-noise-ratios (SNRs) of both sensors are improved with an own analog amplifier module before recording them with an USB oscilloscope. A developed measurement software (LabVIEW) is used to adjust measurement parameters (trigger limit, record length, conversion factors, etc.) and automatically store and separate measurement data from different acoustic events and imprints. To evaluate the result files a second software tool (MATLAB) reads the files out and clusters the recorded events to separate crack signals from electrical and mechanical disturbances. With a prototype first test results are generated and the functionality and accuracy of the measurement concept is proven.

Marangoni flow-guided molecular accumulation for sensitive and rapid SERS detection of phthalates

Public attention has been emerging for the prevention and assessment of healthcare risks from phthalate esters (PAEs) in daily life. For the effective detection of small environmentally hazardous materials, we suggest two major points: i) the formation of high-density hotspots and ii) the delivery of molecules to the activated plasmonic regions. In this study, we developed a method for laser-induced Marangoni flow in cotton fabric (CF) nanopillars (NPs) deposited with Au (Au/CFNPs) for the detection of PAEs by surface-enhanced Raman spectroscopy (SERS). We optimized the performance of the Au/CFNP platforms by adjusting both the maskless plasma etching time and the Au deposition thickness. Narrow (∼8 nm) and populated hotspots were achieved at an Ar plasma treatment time of 30 s and a Au thickness of 150 nm. The molecular behaviors were observed via real-time monitoring of SERS signals. Under continuous laser illumination, Marangoni radial convection flow became predominant over the outward capillary force, resulting in molecular accumulation in the detection areas. For small probe dyes and PAEs, the maximum intensities were achieved within 80 s after the injection of analytes and their signal fluctuations were estimated to have a relative standard deviation of < 10 %, which indicated sensitive and reproducible sensing. The prediction of PAEs extracted from the actual samples was investigated on the basis of a model used to quantitatively fit reference samples and the results were compared with those obtained by high-performance liquid chromatography. The results demonstrated that the combination of densified hotspots on three-dimensional porous substrates and laser-induced Marangoni flow is highly desirable for on-site early screening assays for low-quantity harmful materials present in surrounding environments.

Small molecule probes with strong one and two-photon excited fluorescence for bioimaging

Two-photon excited fluorescence (TPEF) is widely sought after in bioimaging applications due to its exceptional deep tissue penetration and excellent high spatiotemporal resolution. In this paper, benzothiazole and benzimidazole are linked to N, N-diphenylaniline, respectively, through pyridine vinyl. Two new D-π-A (electron donor-π conjugated bridges-electron acceptor) probes (P1, P2) were designed and synthesized. The molecular structures were completely characterized through FT-IR, 1H NMR, 13C NMR, ESI-MS, and X-ray single crystal diffraction. The probes have good one-photon excited fluorescence and two-photon excited fluorescence. Experimental test results indicate that they can be fluorescence probes for biological imaging of HePG2 and juvenile zebrafish. In addition, the fluorescent probes also have the ability to target and locate cancer cell mitochondria.

Mitochondrial Labeling with Mulberrin-Cy3: A New Fluorescent Probe for Live Cell Visualization.

Mitochondria, crucial intracellular organelles, are central to energy metabolism, signal transduction, apoptosis, calcium homeostasis, and a myriad of other biological processes, making them a focal point in diverse research fields. The capacity to fluorescently label and visually track mitochondria is crucial for understanding their biological roles. We present mulberrin-Cy3, a novel small molecule fluorescent probe that selectively labels mitochondria in animal cells, including cancer cells, with relative ease. This protocol details the synthesis of mulberrin-Cy3 and its use for visualizing mitochondria in living cells. The synthesis is straightforward and time-efficient, and the labeling method is more accessible than traditional approaches, providing a cost-effective option for mitochondrial visualization at room temperature. The labeling is rapid, with effective labeling achieved within 5 min of incubation. The fluorescent signal is stable and brighter, offering a significant advantage over existing methods. Mulberrin-Cy3 represents a promising mitochondrial labeling compound, providing researchers with a novel experimental tool to explore the complex biological functions of mitochondria. This innovation has the potential to significantly advance our comprehension of mitochondrial dynamics and their role in cellular health and disease.

Targeting Ras-, Rho-, and Rab-family GTPases via a conserved cryptic pocket

The family of Ras-like GTPases consists of over 150 different members, regulated by an even larger number of guanine exchange factors (GEFs) and GTPase-activating proteins (GAPs) that comprise cellular switch networks that govern cell motility, growth, polarity, protein trafficking, and gene expression. Efforts to develop selective small molecule probes and drugs for these proteins have been hampered by the high affinity of guanosine triphosphate (GTP) and lack of allosteric regulatory sites. This paradigm was recently challenged by the discovery of a cryptic allosteric pocket in the switch II region of K-Ras. Here, we ask whether similar pockets are present in GTPases beyond K-Ras. We systematically surveyed members of the Ras, Rho, and Rab family of GTPases and found that many GTPases exhibit targetable switch II pockets. Notable differences in the composition and conservation of key residues offer potential for the development of optimized inhibitors for many members of this previously undruggable family.

A Nanofluorescent Probe for Evaluating the Fluctuation of Aminopeptidase N in Nonalcoholic Fatty Liver Disease and Hepatic Fibrosis.

Aminopeptidase N (APN/CD13) is a widely expressed transmembrane ectoenzyme that is crucial for maintaining normal physiological activities. It exhibits abnormal activity closely associated with hepatic fibrosis and nonalcoholic fatty liver disease (NAFLD). Therefore, there is a high demand for noninvasive detection of aminopeptidase N (APN) in the diagnosis and research of related diseases. Here, we developed a small molecule fluorescent probe, Hcy-APN, which is a fluorescent probe with high sensitivity and selectivity for the detection of APN. Furthermore, we synthesized the fluorescent nanoprobe Hcy-APN@MSN by self-assembling Hcy-APN and mesoporous silica nanoparticles in solution using a combination of molecular probe design and nanofunctionalization strategies. The detection limit of this probe was 1.5 ng/mL. Hcy-APN@MSN exhibits more stable spectral characteristics compared to Hcy-APN and is suitable for detecting APN activity in live cells and mice. Hcy-APN@MSN was utilized for in vivo and intracellular imaging of NAFLD and hepatic fibrosis at different stages, as well as for a systematic assessment of APN levels in the liver. The results confirm an elevation in the expression levels of APN in NAFLD and hepatic fibrosis models. Furthermore, we investigated the inhibitory effect of the APN inhibitor bestatin in nonalcoholic fatty liver and hepatic fibrosis disease models, confirming its regulatory effect on APN levels in cells and in vivo in both disease models. Therefore, this study may offer diagnostic possibilities for detecting NAFLD and hepatic fibrosis.

Ex Tenebris Lux: Illuminating Reactive Oxygen and Nitrogen Species with Small Molecule Probes.

Reactive oxygen and nitrogen species are small reactive molecules derived from elements in the air─oxygen and nitrogen. They are produced in biological systems to mediate fundamental aspects of cellular signaling but must be very tightly balanced to prevent indiscriminate damage to biological molecules. Small molecule probes can transmute the specific nature of each reactive oxygen and nitrogen species into an observable luminescent signal (or even an acoustic wave) to offer sensitive and selective imaging in living cells and whole animals. This review focuses specifically on small molecule probes for superoxide, hydrogen peroxide, hypochlorite, nitric oxide, and peroxynitrite that provide a luminescent or photoacoustic signal. Important background information on general photophysical phenomena, common probe designs, mechanisms, and imaging modalities will be provided, and then, probes for each analyte will be thoroughly evaluated. A discussion of the successes of the field will be presented, followed by recommendations for improvement and a future outlook of emerging trends. Our objectives are to provide an informative, useful, and thorough field guide to small molecule probes for reactive oxygen and nitrogen species as well as important context to compare the ecosystem of chemistries and molecular scaffolds that has manifested within the field.