Probe Design Strategy Research Articles

Liver injury long-term monitoring and fluorescent image-guided tumor surgery using self-assembly amphiphilic donor-acceptor NIR-II dyes

Real-time monitoring of liver dysfunction represents a significant unmet demand in clinical and preclinical research. The second near-infrared window (NIR-II, 1000–1700 nm) fluorescent imaging is an attractive method for biomedical imaging and may be a promising approach for liver dysfunction monitoring. Herein, we designed and synthesized a small-molecule NIR-II dye TQT 1009 with an asymmetric donor-acceptor (D-A) core. By introducing four kinds of polyethylene glycol (PEG) with different length and molecular weights (nK, n = 0.5, 2, 5, 10) to TQT1009, the dye was self-assembled into different nanoparticles named as TQP nK with regulated size and controllable circulation lifetime in vivo. In general, TQP nK showed a super high contrast ratio for blood vessels, bones, intestines, lymph, and tumor imaging. The best-selected probe, TQP 10K, exhibited ultralong in vivo circulation time (>96 h) which was suitable for long-term quantitative monitoring of liver and vessel function at a single dose, implying the excellent prospects compared with ICG, which was quickly eliminated in blood within a few minutes. Meanwhile, TQP 10K also achieved NIR-II surgical navigation of tumor in an extended time window (>7 d). Overall, our results demonstrate the self-assembly PEGylated amphiphilic TQP nK provide a new probe design strategy for liver function monitoring and image-guided tumor surgery in a prolonged time window.

De novo design of an ultrasensitive fluorogenic probe for aminopeptidase N sensing in living system

Aminopeptidase N is a widely expressed zinc-biding metalloprotease which plays important roles in maintaining normal physiological functions and the abnormal activity of APN is highly related chronic inflammatory disease and cancer progression. Thus, the detection and specific tracing of APN activity is of great importance in clinical diagnosis, drug discovery and disease related biological study. To date, enzyme activated fluorescent probe has attracted intense attention due to its high sensitivity and spatial-temporal resolution and biocompatibility in living system. Many researchers have attempted in constructing APN activity based probes and applicated in medical study such as tumor imaging, photodynamic therapy, and surgical navigation. However, these probes were all acquired by incorporating the fluorophore with its natural substrate. Thus, to discovery a new design strategy for APN targeting could provide efficient alternative method for APN probe construction and APN related chemical biology research. By adapting the unnatural amino-acid (2-Aminobutyric acid, Abu) which showed high affinity and specificity toward APN, we herein proposed a new probe design strategy for APN sensing and a near-infrared fluorescent probe TMN-Abu was constructed through this strategy. The in vitro assays showed that TMN-Abu possess high sensitivity toward APN (LOD = 0.57 ng/mL) and high binding affinity toward APN (Km = 19.1 ± 1.84 μM). Most importantly, TMN-Abu was successfully applicated into monitoring endogenous APN activity in living cells, tumor-bearing nude mice and for the first time applicated into diabetic model mice and APN activity dynamic change in biological events such as starvation and EGF stimulation.

Strategic Design of Amyloid-β Species Fluorescent Probes for Alzheimer's Disease.

Alzheimer's disease (AD) is a high mortality and high disability rates neurodegenerative disease characterized by irreversible progression and poses a significant social and economic burden throughout the world. However, currently approved AD therapeutic agents only alleviate symptoms and there is still a lack of practical therapeutic regimens to stop or slow the progression of this disease. Thus, there is urgently needed novel diagnosis tools and drugs for early diagnosis and treatment of AD. Among several AD pathological hallmarks, amyloid-β (Aβ) peptide deposition is considered a critical initiating factor in AD. In recent years, with the advantages of excellent sensitivity and high resolution, near-infrared fluorescence (NIRF) imaging has attracted the attention of many researchers to develop Aβ plaque probes. This review mainly focused on different NIRF probe design strategies for imaging Aβ species to pave the way for the future design of novel NIRF probes for early diagnosis AD.

Photo-Brook rearrangement of acyl silanes as a strategy for photoaffinity probe design.

Photoaffinity labeling (PAL) is a powerful tool for the identification of non-covalent small molecule–protein interactions that are critical to drug discovery and medicinal chemistry, but this approach is limited to only a small subset of robust photocrosslinkers. The identification of new photoreactive motifs capable of covalent target capture is therefore highly desirable. Herein, we report the design, synthesis, and evaluation of a new class of PAL warheads based on the UV-triggered 1,2-photo-Brook rearrangement of acyl silanes, which hitherto have not been explored for PAL workflows. Irradiation of a series of probes in cell lysate revealed an iPr-substituted acyl silane with superior photolabeling and minimal thermal background labeling compared to other substituted acyl silanes. Further, small molecule (+)-JQ1- and rapamycin-derived iPr acyl silanes were shown to selectively label recombinant BRD4-BD1 and FKBP12, respectively, with minimal background. Together, these data highlight the untapped potential of acyl silanes as a novel, tunable scaffold for photoaffinity labeling.

Magnetic Resonance Imaging of PSMA-Positive Prostate Cancer by a Targeted and Activatable Gd(III) MR Contrast Agent.

Prostate-specific membrane antigen (PSMA) is a transmembrane protein that is highly expressed in aggressive prostate cancer (PCa) and has been extensively studied as a PCa diagnostic imaging biomarker. Multiple imaging modalities have exploited PSMA as a biomarker including magnetic resonance (MR), Optical, and PET imaging. Of all the imaging MR imaging provides the most detailed information, concurrently providing anatomical, functional, and potentially molecular information. However, the lower sensitivity of MR requires development of molecular MR contrast agents that provides high signal-to-noise ratios. Herein, we report the first targeted and activatable Gd(III)-based MR contrast agents prostate cancer probe 1 and 2 (PCP-1 and -2). We successfully used PCP-2 to differentiate between PSMA+ and PSMA- prostate cancer cells with both in vitro fluorescence imaging and in vivo MR imaging. The in vivo MR imaging results were further supported by ex vivo fluorescence imaging studies, showcasing the unique bimodal feature of PCP-2. Furthermore, PCP-2 highlights a unique molecular MR probe design strategy that improved the sensitivity of traditional biomarker-targeted MR imaging, addressing a critical unmet need in molecular MR imaging field. This work represents the first example of a targeted and activatable MR contrast agent that can be systemically administered in vivo to highlight PSMA+ prostate tumors, paving the way for the clinical translation of MR PSMA imaging.

Strategy of De Novo Design toward First-In-Class Imaging Agents for Simultaneously Differentiating Glioma Boundary and Grades.

The extent of resection and tumor grade are two predominant prognostic factors for glioma. Fluorescent imaging is promising to facilitate accurate resection and simultaneous tumor grading. However, no probe fulfilling this task has been reported. Herein, we proposed a strategy of de novo design toward first-in-class fluorescent probes for simultaneously differentiating glioma boundary and grades. By bioinformatics analysis in combination with experimental validation, platelet-derived growth factor receptor β (PDGFRβ) was revealed as a promising biomarker for glioma imaging and grading. Then, fluorogenic probe PDGFP 1 was designed, guided by the structure-activity relationship study. Finally, the probe was demonstrated to stain glioma cells and tissues in the mice orthotopic glioma model with high selectivity over normal brain cells or tissues. Meanwhile, ex vivo experiments using patient-derived samples indicated that the fluorescence was significantly positively correlated with the tumor grades. This result highlighted the feasibility of the three-step de novo probe design strategy and suggested PDGFP 1 as a promising probe for simultaneously differentiating glioma boundary and grades, showing prospects of clinical translation.

A fluorescent probe with dual acrylate sites for discrimination of different concentration ranges of cysteine in living cells

Monitoring of cysteine (Cys) is of significant importance for studying Cys-involved biological functions and clinically diagnosing Cys-related diseases. Recently, few fluorescent probes with two different reacting sites were reported to be capable of sensing different concentration ranges of Cys with distinct fluorescence signals, particularly suiting for bioimaging. However, due to relative sophisticated synthesis and moderate selectivity, the applications of these probes were still severely restricted. In this work, we proposed a novel probe design strategy by utilizing two same reacting groups, instead of two different reacting groups, to simplify the synthesis route and minimize the interference from competing species. Same reacting groups in a probe with different steric hindrances could exhibit different reactivities to Cys. This probe showed distinguishable fluorescence peak wavelengths towards low and high concentration ranges of Cys, giving green and blue emissions, respectively. Moreover, this probe was successfully applied for monitoring of Cys concentration in living cells. We believe this work provided a simpler strategy for dual-site fluorescent probes to sense difference concentration ranges of Cys, which may inspire more probe design in future.

Bioinspired Design of Reversible Fluorescent Probes for Tracking Nitric Oxide Dynamics in Live Cells

Bioinspired Design of Reversible Fluorescent Probes for Tracking Nitric Oxide Dynamics in Live Cells

Design Strategy of Fluorescent Probes for Live Drug-Induced Acute Liver Injury Imaging

Drug-induced acute liver injury (DIALI) is increasingly recognized as a significant cause of acute liver injury (ALI), which is characterized by a rapid loss of hepatocyte function in patients without pre-existing liver diseases. Evaluation of corresponding biomarkers, including alanine transaminase and aspartate amino transferase, is available as a diagnostic tool for hepatotoxicity. However, these blood tests have certain limitations: (1) they are generally not available for early estimation; (2) it is difficult to visualize and identify hepatotoxicity unambiguously in real-time; and (3) the biomarkers are not unique and are usually influenced by a variety of diseases, leading to potential false results. It is of grave importance and burgeoning demand to develop an early diagnostic approach for such diseases, but the ideal toolkit remains an unresolved challenge.As an alternative, molecular optical probes (fluorescence, chemiluminescence, bioluminescence, etc.) display a lot of advantages, such as high sensitivity, noninvasive fast analysis, and real-time in situ detection. They have emerged as potent and promising tools for the biomedical study of DIALI in living system. Until now, a number of optical probes for DIALI have been reported with some great potential for clinical trials. However, most of the probes still suffer from false signals because of the limitations in clinical application, including poor selectivity, low sensitivity, and biocompatibility. One key challenge that probes face in the ALI environment is the excessive exposure to reactive oxygen/nitrogen species and diffusivity, which may lead to false-positive or negative signals.Our group has employed multiple rational approaches to engineer high-performance optical probes for DIALI. With such development, we have successfully achieved the accurate detection of DIALI with minimal false signals both ex vivo and in vivo. While marching firmly toward understanding the biogenesis and progression of DIALI, we ultimately aim at the early stage clinical diagnosis of the disease, as well as mechanism understanding for clinical trials. In this Account, we summarize and present our three new approaches for the development of high-fidelity optical probes: (1) a combined screening and rational design strategy, (2) a double-locked probe design strategy, and (3) in situ imaging based on the release of a precipitating fluorochrome strategy. Using these strategies, we have formulated probes for a range of biological species that are biomarkers of DIALI, including reactive nitrogen species (ONOO-), reactive sulfur species (H2S and H2Sn), and enzymes (LAP, MAO, and ALP). We have highlighted the rationale for our design and screening strategy and methods to achieve high-fidelity optical probes. Some recent examples of optical probes developed by our laboratory and collaborations are mainly illustrated herein. We anticipate the strategies summarized here to inspire future molecular optical probe design, to contribute to studies of the detailed molecular mechanisms underlying liver diseases, and to improve the efficiency of the diagnosis and treatment of these diseases in clinical settings.

Ultrasensitive, Specific, and Rapid Fluorescence Turn-On Nitrite Sensor Enabled by Precisely Modulated Fluorophore Binding.

The precise regulation of fluorophore binding sites in an organic probe is of great significance toward the design of fluorescent sensing materials with specific functions. In this study, a probe with specific fluorescence properties and nitrite detection ability is designed by precisely modulating benzothiazole binding sites. Only the fluorophore bond at the ortho‐position of the aniline moiety can specifically recognize nitrite, which ensures that the reaction products displays a robust green emission. The unique 2‐(2‐amino‐4‐carboxyphenyl) benzothiazole (ortho‐BT) shows superior nitrite detection performance, including a low detection limit (2.2 fg), rapid detection time (<5 s), and excellent specificity even in the presence of >40 types of strong redox active, colored substances, nitro compounds, and metal ions. Moreover, the probe is highly applicable for the rapid on‐site and semiquantitative measurement of nitrite. The proposed probe design strategy is expected to start a new frontier for the exploration of probe design methodology.

Next-generation Viral RNA/DNA in situ Hybridization Applications in Human Immunodeficiency Virus/Simian Immunodeficiency Virus Research.

In situ hybridization is a powerful technique to identify specific RNA or DNA sequences within individual cells in tissue sections, providing important insights into physiological processes and disease pathogenesis. In situ hybridization (ISH) has been used for many years to assess the location of cells infected by viruses, but recently a next-generation ISH approach was developed with a unique probe design strategy that allows simultaneous signal amplification and background suppression to achieve single-molecule visualization while preserving tissue morphology. This next-generation ISH is based on an approach like branched PCR, but performed in situ and is more facile, sensitive, and reproducible than classical ISH methods or in situ PCR approaches in routinely detecting RNA or DNA in formalin-fixed paraffin embedded (FFPE) tissues. For the last several years our laboratory has been applying this ISH platform for the detection of human immunodeficiency (HIV) and simian immunodeficiency (SIV) viral RNA (vRNA) and/or viral DNA (vDNA) positive cells within a multitude of FFPE tissues. With this detailed technical manuscript, we would like to share our knowledge and advice with all individuals interested in using next-generation ISH in their research.

A facile AIEgen-based fluorescent probe design strategy and its application in hypochlorite probe construction

Fluorogens with aggregation-induced emission (AIE) characteristics (AIEgens) and the strategy for constructing AIEgen-based fluorescent probes have attracted extensive attention. In this work, inspired by the relationship between chemical structures and fluorescence properties of three tetraphenylethylene (TPE)-based compounds, a facile strategy by modifying of AIEgen with electron-donating substituent (EDS) and electron-withdrawing substituent (EWS) capable of reacting with target for fluorescent probe design was proposed. Under the guidance, a series of hypochlorite (ClO−) probes named TPEP-CN, TPEP-NO2, M-TPEP-CN and M-TPEP-NO2 were rationally designed and synthesized, and their sensing performances were studied. Finally, M-TPEP-CN with the most obvious fluorescence color change from orange to green in the presence of ClO− was selected, and then successfully applied to ClO− detection in aqueous solution as well as the construction of portable test strips and cell imaging, which confidently proved the feasibility of our probe design strategy and more excellent probes will be developed according to this strategy in the future.

Robust Organoalkoxysilanes as Red Unconventional Fluorescent Platform

AbstractUnconventional fluorescent materials have attracted intense and continuous attention due to the facile processability, excellent biocompatibility, and high availability. However, for the lack of suitable unconventional fluorescent platform, unconventional luminophore‐based fluorescent probes have not been applied in the biological field, especially in the detection of bioactive molecules. In this work, unconventional red fluorescence is observed from a series of organoalkoxysilanes for the first time. Particularly, the unique fluorescence derived from smart Si–O bridged structures prompt the fluorescent probe design strategy. The strategy involves applying the Si–O bridge to provide desirable red unconventional fluorescence, and ratiometric detection of endogenous nitric oxide in lysosomes and in vivo. It is expected that this novel strategy will expand the applications of unconventional fluorescence to the bioimaging field, and further provide valid approach for the future evolution of unconventional fluorescent probes.

Sequentially Activated Probe Design Strategy for Analyzing Metabolite Crosstalk in a Biochemical Cascade.

Interrogating metabolite crosstalk in live cells is important to understand the interplay between metabolic and signal transduction pathways but is challenging due to the lack of efficient analytical techniques. Here we report a sequentially activated probe design strategy resulting in probe HF-6 being capable of imaging the crosstalk between H2O2 and formaldehyde in live cells. Fluorescence of HF-6 can only be triggered by first H2O2 activation followed by binding with formaldehyde. Facilitated by this sequentially activated mechanism, HF-6 imaging revealed H2O2-induced upregulation of formaldehyde in live SH-SY5Y cells, while little change of intracellular H2O2 level was observed when cells were stimulated with formaldehyde for limited time. These results establish a link for the crosstalk between H2O2 and formaldehyde in redox signaling and provide a starting point to study broader metabolite interactions.

Modular, triple-resonance, transmission line DNP MAS probe for 500 MHz/330 GHz.

We describe the design and construction of a modular, triple-resonance, fully balanced, DNP-MAS probe based on transmission line technology and its integration into a 500 MHz/330 GHz DNP-NMR spectrometer. A novel quantitative probe design and characterization strategy is developed and employed to achieve optimal sensitivity, RF homogeneity and excellent isolation between channels. The resulting three channel HCN probe has a modular design with each individual, swappable module being equipped with connectorized, transmission line ports. This strategy permits attachment of a mating connector that facilitates accurate impedance measurements at these ports and allows characterization and adjustment (e.g. for balancing or tuning/matching) of each component individually. The RF performance of the probe is excellent; for example, the 13C channel attains a Rabi frequency of 280 kHz for a 3.2 mm rotor. In addition, a frequency tunable 330 GHz gyrotron operating at the second harmonic of the electron cyclotron frequency was developed for DNP applications. Careful alignment of the corrugated waveguide led to minimal loss of the microwave power, and an enhancement factor ε = 180 was achieved for U-13C urea in the glassy matrix at 80 K. We demonstrated the operation of the system with acquisition of multidimensional spectra of cross-linked lysozyme crystals which are insoluble in glycerol-water mixtures used for DNP and samples of RNA.

Nucleic-Acid Structures as Intracellular Probes for Live Cells.

The chemical composition of cells at the molecular level determines their growth, differentiation, structure, and function. Probing this composition is powerful because it provides invaluable insight into chemical processes inside cells and in certain cases allows disease diagnosis based on molecular profiles. However, many techniques analyze fixed cells or lysates of bulk populations, in which information about dynamics and cellular heterogeneity is lost. Recently, nucleic-acid-based probes have emerged as a promising platform for the detection of a wide variety of intracellular analytes in live cells with single-cell resolution. Recent advances in this field are described and common strategies for probe design, types of targets that can be identified, current limitations, and future directions are discussed.

Advances in detection of nucleic acid tumor biomarkers via electrochemical biosensors

The detection of tumor biomarkers can result in early diagnosis and monitoring of the development and prognosis of cancer. With the development of high-throughput sequencing technology, various types of nucleic acids have been identified to be associated with tumorigenesis and tumor progression. These nucleic acids can be used as tumor biomarkers and play an important role in the early diagnosis of cancer. Because most nucleic acids have the characteristics of instability, dynamic expression, and low abundance, it is particularly important to detect these types of tumor markers quickly, sensitively, and specifically. Electrochemical biosensor technology is a fast-developing method that meets these requirements. Compared with the traditional detection methods, electrochemical detection has attracted more attention due to its high efficiency, sensitivity, and simplicity. Moreover, the detection equipment is portable, cheap, and easily miniaturized and integrated. Recently, electrochemical biosensors for nucleic acid detection have made considerable advances and have demonstrated their potential application in precision medicine. This review focuses on various types of nucleic acid tumor biomarkers and summarizes the latest progress achieved in recent years in developing electrochemical sensors for detecting nucleic acid markers, with an emphasis on the probe design and signal amplification strategies in electrochemical sensor systems.

Nanoamplicon Comparator for Live-Cell MicroRNA Imaging.

As an investigative tool, live-cell imaging requires superior probe design to guarantee imaging quality and data validity. The ability to simultaneously address the robustness, sensitivity, and consistency issues in a single-assay system is highly desired, but it remains a largely unsolved challenge. We describe herein a probe-design strategy called a nanoamplicon comparator (NAC) and demonstrate its proof-of-concept utility in intracellular microRNA (miRNA) imaging. This novel designer architecture builds upon spherical nucleic acids (SNAs) for robustness, catalytic hairpin assembly (CHA) for sensitivity, and upconversion nanoparticles (UNPs) for consistency. A catalytic circuit comprising a UNP-hairpin-DNA (UNP-HDNA) conjugate and a hairpin-DNA-organic-fluorophore (HDNA-F) conjugate as probe responds to target miRNA and generates the UNP-HDNA-HDNA-F complex as an NAC for quantitative UNP-to-organic-fluorophore-luminescence-resonance-energy-transfer (LRET) imaging against a native UNP-emission reference channel. An imaging application with miR21 shows the ability to monitor miRNA-expression levels across different cell lines and under an external stimulus.

GOPHER: Generator Of Probes for capture Hi-C Experiments at high Resolution

BackgroundTarget enrichment combined with chromosome conformation capturing methodologies such as capture Hi-C (CHC) can be used to investigate spatial layouts of genomic regions with high resolution and at scalable costs. A common application of CHC is the investigation of regulatory elements that are in contact with promoters, but CHC can be used for a range of other applications. Therefore, probe design for CHC needs to be adapted to experimental needs, but no flexible tool is currently available for this purpose.ResultsWe present a Java desktop application called GOPHER (Generator Of Probes for capture Hi-C Experiments at high Resolution) that implements three strategies for CHC probe design. GOPHER’s simple approach is similar to the probe design of previous approaches that employ CHC to investigate all promoters, with one probe being placed at each margin of a single digest that overlaps the transcription start site (TSS) of each promoter. GOPHER’s simple-patched approach extends this methodology with a heuristic that improves coverage of viewpoints in which the TSS is located near to one of the boundaries of the digest. GOPHER’s extended approach is intended mainly for focused investigations of smaller gene sets. GOPHER can also be used to design probes for regions other than TSS such as GWAS hits or large blocks of genomic sequence. GOPHER additionally provides a number of features that allow users to visualize and edit viewpoints, and outputs a range of files useful for documentation, ordering probes, and downstream analysis.ConclusionGOPHER is an easy-to-use and robust desktop application for CHC probe design. Source code and a precompiled executable can be downloaded from the GOPHER GitHub page at https://github.com/TheJacksonLaboratory/Gopher.

Recognition-Enhanced Metastably Shielded Aptamer for Digital Quantification of Small Molecules.

Aptamers are recognized as competitive affinity reagents; their application, however, often suffers from their relatively low target binding affinity, especially for small molecules. We herein introduce the concept of a recognition-enhanced metastably shielded aptamer probe (RMSApt) and explore its performance for digital quantification of low-affinity small molecules. The RMSApt design employs the idea of constructing an allosteric aptamer probe conferring a minor energy gap in the recognition switch process to facilitate target binding and probe response, in turn significantly improving the recognition efficiency for low-affinity targets. The probe design strategy boosts the application of aptamers for precisely quantifying targets with a dissociation constant Kd ranging from 10-4 to 10-9 M, which would cover most of the small-molecule species that exist binding aptamers. Thus, RMSApt would facilitate the translation of aptamers for medical diagnosis, food safety, and environmental screening.