Low Background Signal Research Articles

Abstract 7021: Selecting your epigenomic assay: Comparing ChIP-seq, CUT&RUN, and CUT&Tag

Abstract For many years, chromatin immunoprecipitation (ChIP) was the standard assay for studying epigenetic mechanisms. However, two new versatile techniques have recently been developed that are faster, experimentally easier, and more cost-effective than ChIP. Cleavage Under Targets & Release Using Nuclease (CUT&RUN) and Cleavage Under Targets & Tagmentation (CUT&Tag) can be used for understanding protein-DNA interactions within the natural chromatin context of the cell. Both CUT&RUN and CUT&Tag can be combined with Next Generation Sequencing (NGS) to analyze the function of histone modifications and the binding of transcription factors, DNA replication factors, or DNA repair proteins across the genome. CUT&RUN and CUT&Tag are rapid, robust, and true low cell number methods for the detection of protein-DNA interactions. Unlike ChIP, both assays are free of formaldehyde cross-linking, chromatin fragmentation, and immunoprecipitation. When compared to ChIP, CUT&RUN and CUT&Tag require fewer starting cells (as low as 5,000 – 20,000), are shorter assays (one day from cells to DNA), and generate lower background signal (requiring fewer sequencing reads). At Cell Signaling Technology (CST), we have developed ChIP-seq, CUT&RUN, and CUT&Tag assay kits and validated a broad set of antibodies against histone modifications, transcription factors, and cofactors that are compatible with these kits. This poster will discuss the fundamentals of all three assays and highlight important factors to consider when selecting an assay for your experiment. Through our antibody validation efforts, we have also generated a unique and extensive archive of ChIP-seq, CUT&RUN, and CUT&Tag NGS data using recombinant, monoclonal antibodies. This allows for the controlled comparison of these three commonly used chromatin profiling techniques. In this poster, I will highlight the similarities and differences between these assays when mapping a variety of histone modifications, transcription factors, and cofactor binding events across multiple sample types. Citation Format: Angela H. Guo, Christopher R. Comeau, Fang Chen. Selecting your epigenomic assay: Comparing ChIP-seq, CUT&RUN, and CUT&Tag [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7021.

Colorimetric detection of Staphylococcus aureus based on self-linkable and hollow organic-inorganic nanocomposites as light-driven oxidase-like nanozyme for cascade signal amplification

Facing increasingly serious bacterial infections and their threat to food safety and human health, exploring specific and sensitive bacterial strategies is indispensable. Nanozyme-based colorimetric biosensors with the superiorities of easy-to-read and catalytic signal amplification have attracted increasing interest. However, few investigations relate to the nanozymes with excellent light-driven oxidase-like activity. In this study, a sensitive colorimetric biosensor for Staphylococcus aureus (S. aureus) detection was constructed based on a light-driven oxidase-like nanozyme (PHCS@CuNcPOPTP) as well as the self-chain linking process. The hollow organic-inorganic nanocomposite (PHCS@CuNcPOPTP) was prepared by a one-step Friedel-Crafts alkylation reaction. The PHCS@CuNcPOPTP exhibited excellent and synergistically enhanced light-driven oxidase-like activity originating from its unique photosensitive properties. Subsequently, PHCS@CuNcPOPTP was served as a scaffold, loading with the rabbit anti-Staphylococcus aureus Rosenbach tropina antibody (bs-4582R, Ab2), DNA1, and DNA2, respectively, to construct signal probes (PHCS@CuNcPOPTP@Au-Ab2/DNA1, PHCS@CuNcPOPTP@Au-DNA1, and PHCS@CuNcPOPTP@Au-DNA2). By the repetitive DNA hybridization among signal probes, a large amount of PHCS@CuNcPOPTP-DNA dendrimers were formed and utilized to trigger the chromogenic reaction without the participation of high-concentration H2O2, resulting in the cascaded catalytic amplification and the lower background signal. As expected, the suggested sensing platform presented ultrasensitive bacterial detection with a remarkably broad linear range (101 to 108 CFU/mL) and a limit of detection (LOD, 3.40 CFU/mL).

Surfactant-based supramolecular dye assembly: A highly selective and economically viable platform for quantification of heparin antidote

Herein, we have employed a supramolecular assembly of a cationic dye, LDS-698 and a common surfactant sodium dodecyl sulfate (SDS) as a turn-on fluorescent sensor for protamine (Pr) detection. Addition of cationic Pr to the solution of dye-surfactant complex brings negatively charged SDS molecules together through strong electrostatic interaction, assisting aggregation of SDS way before its critical micellar concentration (CMC). These aggregates encapsulate the dye molecules within their hydrophobic region, arresting non-radiative decay channels of the excited dye. Thus, the LDS-698•SDS assembly displays substantial enhancement in fluorescence intensity that follows a nice linear trend with Pr concentration, providing limit of detection (LOD) for Pr as low as 3.84(±0.11) nM in buffer, 124.4(±6.7) nM in 1% human serum and 28.3(±0.5) nM in 100% human urine. Furthermore, high selectivity, low background signal, large stokes shift, and emission in the biologically favorable deep-red region make the studied assembly a promising platform for Pr sensing. As of our knowledge it is the first ever Pr sensory platform, using a very common surfactant (SDS), which is economically affordable and very easily available in the market. This innovative approach can replace the expensive, exotic and specialized chemicals considered for the purpose and thus showcase its potential in practical applications.

Development of highly sensitive dual-enhanced fluorescence quenching immunochromatographic test strips based on Pt nanoprobes

The fluorescence-quenching method is crucial in vitro analysis, particularly for immunochromatographic test strips (ICTs) using noble metal nanoparticles as probes. However, ICTs still fall short in meeting the requirements for the detection of traces biomarkers due to the noble metal nanoparticles can only quench fluorescence of the dyes within a confined distance. Interestingly, noble metal nanoparticles, such as Pt NPs cannot only perform fluorescence-quenching ability based on the Förster resonance energy transfer (FRET), but also show perfect oxidase-like catalytic performance on many kinds of substrates, such as 3,3’,5,5’ -tetramethylbenzidine (TMB). We observed that the oxTMB (the oxidation products of TMB) exhibited notable effectiveness in quenching Cy5 fluorescence by the strong inner filter effect (IFE), which obviously improved the fluorescence-quenching efficiency with extremely low background signal. Through the dual-enhanced fluorescence quenching mechanism, the fluorescence quenching constant (Kn) was 661.24-fold that of only Pt NPs on the NC membrane. To validate the feasibility of this technique, we employed two types of biomarkers, namely microRNA (miR-15a-5p) and the signature protein (PSA). The sensitivity of miR-15a-5p was 9.286 × 10−18 mol/L and 17.5-fold more than that based on Pt NPs. As for the PSA, the LOD (0.6265 pg/mL) was 15.5-fold enhancement more sensitive after catalysis. Overall, the dual-enhanced fluorescence quenching rFICTs could act as a practical detection for biomarker in real samples.

Using Cleavage Under Targets and Tagmentation (CUT&Tag) Assay in Mouse Myoblast Research.

This protocol paper aims to provide the new researchers with the full details of using Cleavage Under Targets and Tagmentation (CUT&Tag) to profile the genomic locations of chromatin binding factors, histone marks, and histone variants. CUT&Tag protocols function very well with mouse myoblasts and freshly isolated muscle stem cells (MuSCs). They can easily be applied to many other cell types as long as the cells can be immobilized by Concanavalin-A beads. Compared to CUT&Tag, chromatin immunoprecipitation (ChIP) assays are time-consuming experiments. ChIP assays require the pre-treatment of chromatin before the chromatic material can be used for immunoprecipitation. In cross-linking ChIP (X-ChIP), pre-treatment of chromatin involves cross-linking and sonication to fragment the chromatin. In the case of native ChIP (N-ChIP), the fragmented chromatins are normally achieved by Micrococcal nuclease (MNase) digestion. Both sonication and MNase digestion introduce some bias to the ChIP experiments. CUT&Tag assays can be finished within fewer steps and require much fewer cells compared to ChIPs but provide more unbiased information on transcription factors or histone marks at various genomic locations. CUT&Tag can function with as few as 5,000 cells. Due to its higher sensitivity and lower background signal than ChIPs, researchers can expect to obtain reliable peak data from merely several millions of reads after sequencing.

Radiolabeling Diaminosarcophagine with Cyclotron-Produced Cobalt-55 and [55Co]Co-NT-Sarcage as a Proof of Concept in a Murine Xenograft Model.

Cobalt-sarcophagine complexes exhibit high kinetic inertness under various stringent conditions, but there is limited literature on radiolabeling and in vivo positron emission tomography (PET) imaging using no carrier added 55Co. To fill this gap, this study first investigates the radiolabeling of DiAmSar (DSar) with 55Co, followed by stability evaluation in human serum and EDTA, pharmacokinetics in mice, and a direct comparison with [55Co]CoCl2 to assess differences in pharmacokinetics. Furthermore, the radiolabeling process was successfully used to generate the NTSR1-targeted PET agent [55Co]Co-NT-Sarcage (a DSar-functionalized SR142948 derivative) and administered to HT29 tumor xenografted mice. The [55Co]Co-DSar complex can be formed at 37 °C with purity and stability suitable for preclinical in vivo radiopharmaceutical applications, and [55Co]Co-NT-Sarcage demonstrated prominent tumor uptake with a low background signal. In a direct comparison with [64Cu]Cu-NT-Sarcage, [55Co]Co-NT-Sarcage achieved a higher tumor-to-liver ratio but with overall similar biodistribution profile. These results demonstrate that Sar would be a promising chelator for constructing Co-based radiopharmaceuticals including 55Co for PET and 58mCo for therapeutic applications.

Peptidoglycan-Targeted [18F]3,3,3-Trifluoro-d-alanine Tracer for Imaging Bacterial Infection.

Imaging is increasingly used to detect and monitor bacterial infection. Both anatomic (X-rays, computed tomography, ultrasound, and MRI) and nuclear medicine ([111In]-WBC SPECT, [18F]FDG PET) techniques are used in clinical practice but lack specificity for the causative microorganisms themselves. To meet this challenge, many groups have developed imaging methods that target pathogen-specific metabolism, including PET tracers integrated into the bacterial cell wall. We have previously reported the d-amino acid derived PET radiotracers d-methyl-[11C]-methionine, d-[3-11C]-alanine, and d-[3-11C]-alanine-d-alanine, which showed robust bacterial accumulation in vitro and in vivo. Given the clinical importance of radionuclide half-life, in the current study, we developed [18F]3,3,3-trifluoro-d-alanine (d-[18F]-CF3-ala), a fluorine-18 labeled tracer. We tested the hypothesis that d-[18F]-CF3-ala would be incorporated into bacterial peptidoglycan given its structural similarity to d-alanine itself. NMR analysis showed that the fluorine-19 parent amino acid d-[19F]-CF3-ala was stable in human and mouse serum. d-[19F]-CF3-ala was also a poor substrate for d-amino acid oxidase, the enzyme largely responsible for mammalian d-amino acid metabolism and a likely contributor to background signals using d-amino acid derived PET tracers. In addition, d-[19F]-CF3-ala showed robust incorporation into Escherichia coli peptidoglycan, as detected by HPLC/mass spectrometry. Based on these promising results, we developed a radiosynthesis of d-[18F]-CF3-ala via displacement of a bromo-precursor with [18F]fluoride followed by chiral stationary phase HPLC. Unexpectedly, the accumulation of d-[18F]-CF3-ala by bacteria in vitro was highest for Gram-negative pathogens in particular E. coli. In a murine model of acute bacterial infection, d-[18F]-CF3-ala could distinguish live from heat-killed E. coli, with low background signals. These results indicate the viability of [18F]-modified d-amino acids for infection imaging and indicate that improved specificity for bacterial metabolism can improve tracer performance.

Exploring T7 RNA polymerase-assisted CRISPR/Cas13a amplification for the detection of BNP via electrochemiluminescence sensing platform

Brain natriuretic peptide (BNP) is considered to be an important biomarker of heart failure (HF) attracting attention. However, its low concentration and short half-life in blood lead to a low-sensitivity detection of BNP, which is a challenge that has to be overcome. In this work, we propose a highly specific, highly sensitive T7 RNA polymerase-assisted clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system to detect BNP via an electrochemiluminescence (ECL) sensing platform and incorporate exonuclease III (Exo III)-hairpin and dumbbell-shaped hybridization chain reaction (HCR) technologies. In this detection scheme, the ECL sensing platform possesses low background signal and high sensitivity. Firstly, the T7 promoter-initiated T7 RNA polymerase acts as a signal amplification technique to generate large amounts of RNAs that can activate CRISPR/Cas13a activity. Secondly, CRISPR/Cas13a is able to trans-cleave the surrounding trigger strand to produce DNA1. Thirdly, DNA1 is involved in the co-amplification reaction of Exo III and hairpin DNA, which subsequently triggers a dumbbell-shaped HCR technology. Eventually, a large number of Ru (II) molecules are inserted into the interstitial space of the dumbbell-shaped HCR to generate a strong ECL signal. The CRISPR/Cas13a possesses outstanding specificity for a single base and increased sensitivity. The tightly conformed dumbbell-shaped HCR provides higher sensitivity than the traditional linear HCR amplification technique. Ultimately, the clever combination of several amplification reactions enables the limit of detection (LOD) as low as 3.2 fg/mL. It showed promise for clinical sample testing, with recovery rates ranging from 98.4% to 103% in 5% human serum samples. This detection method offered a valuable tool for early HF detection, emphasizing the synergy of amplification strategies and specificity conferred by CRISPR/Cas13a technology.

Construction of a Cu2+-Responsive NIR Fluorescent Probe and the Preliminary Evaluation of its Multifunctional Application.

Cu2+ was deemed as toxic and the most common heavy metal pollution in the water and food. Meanwhile, endogenous Cu2+ was deeply involved in plenty of physiological and pathological processes of human. Cu2+ imbalance was related to multiple diseases. Here we developed a Cu2+-responsive NIR probe HX, which not only demonstrated obvious color change when subjected to Cu2+, but also showed linear-dependent NIR fluorescence emission to Cu2+ concentration for Cu2+ detection and quantification both in vitro and in vivo. When HX was applied to imaging Cu2+ in the cell or living animals, intracellular Cu2+ fluctuation and Cu2+ accumulation in the liver could be visualized to indicate the copper level in the cell or organs with low background signals. Meanwhile, by applying HX to monitor Cu2+ uptake in the tumor, copper transporter function could be evaluated to screen the patient who are sensitivity to platinum drug.

Metal–organic frameworks with aggregation‐induced emission

AbstractAggregation‐induced emission (AIE) materials exhibit remarkable emission in the aggregated or solid state while demonstrating minimal emission in dilute solutions. In contrast to conventional luminescent materials, AIE luminogens (AIEgens) offer several advantages in the aggregate state, including high quantum yield, excellent photostability, and low background signals, making them highly promising for diverse applications. Integrating AIEgens into designable metal–organic frameworks (MOFs) enables tunable and well‐ordered AIE materials, allowing for precise control over photophysical properties and deeper exploration of AIE mechanisms. Numerous AIE MOFs have been constructed and investigated, and several reviews focus on their structure design and applications in sensing and bioimaging. This review highlights the state‐of‐the‐art advancements in AIE MOFs, including mechanisms, design strategies, and applications in chemical sensing, bioimaging, and disease therapy. The challenges associated with practical applications of AIE MOFs are also addressed, with an emphasis on their large‐scale production involved interdisciplinary collaboration. This comprehensive review aims at guiding further development of AIE MOFs and promoting their practical applications in analysis, healthcare, and other luminescence related fields.

ZIF-67 Anchored on MoS2/rGO Heterostructure for Non-Enzymatic and Visible-Light-Sensitive Photoelectrochemical Biosensing.

Graphene and graphene-like two-dimensional layered nanomaterials-based photoelectrochemical (PEC) biosensors have recently grown rapidly in popularity thanks to their advantages of high sensitivity and low background signal, which have attracted tremendous attention in ultrahigh sensitive small molecule detection. This work proposes a non-enzymatic and visible-light-sensitive PEC biosensing platform based on ZIF-67@MoS2/rGO composite which is synthesized through a facile and one-step microwave-assisted hydrothermal method. The combination of MoS2 and rGO could construct van der Waals heterostructures, which not only act as visible-light-active nanomaterials, but facilitate charge carriers transfer between the photoelectrode and glassy carbon electrode (GCE). ZIF-67 anchored on MoS2/rGO heterostructures provides large specific surface areas and a high proportion of catalytic sites, which cooperate with MoS2 nanosheets, realizing rapid and efficient enzyme-free electrocatalytic oxidation of glucose. The ZIF-67@MoS2/rGO-modified GCE can realize the rapid and sensitive detection of glucose at low detection voltage, which exhibits a high sensitivity of 12.62 μAmM-1cm-2. Finally, the ZIF-67@MoS2/rGO PEC biosensor is developed by integrating the ZIF-67@MoS2/rGO with a screen-printed electrode (SPE), which exhibits a high sensitivity of 3.479 μAmM-1cm-2 and a low detection limit of 1.39 μM. The biosensor's selectivity, stability, and repeatability are systematically investigated, and its practicability is evaluated by detecting clinical serum samples.

Fluorescent aptasensor for highly sensitive detection of Staphylococcus aureus based on dual-amplification strategy by integrating DNA walking and hybridization chain reaction

Food-borne diseases caused by bacteria threaten human health. Herein, we presented a new fluorescent aptasensor by coupling DNA walking and hybridization chain reaction (HCR) for convenient and sensitive quantification of bacteria. Staphylococcus aureus (S. aureus) was selected as target. When there was target in the system, the binding of S. aureus with its aptamer caused the disintegration of aptamer/DNA walker on the surface of AuNPs and released DNA walker. With the help of Nt.BsmAI, DNA walker moved along the surface of AuNPs and trigger probe was detached from AuNPs. The trigger probe could initiate hybridization chain reaction (HCR) and opened the stems of H1@AuNPs probe and H2@AuNPs probe. After the addition of nicking endonuclease, the adjacent upconversion nanoparticles (UCNPs, NaYF4:Yb3+, Er3+) were further away from the quenchers (AuNPs) of H1 and H2. Therefore, the fluorescence intensity of UCNPs could be restored via fluorescence resonance energy transfer (FRET). Bacteria were thus detected by recording the fluorescence intensity of UCNPs. This method is simple, rapid and sensitive. It can directly detect bacteria in a low background signal. The limit of detection (LOD) was 10 CFU/mL, detection time was less than 3 h. Recovery rates in simulated milk, honey and human serum samples ranged from 93.6 % to 105.8 %. The strategy opens up new paths for early diagnosis of diseases and food monitoring.

Label-free and highly sensitive detection of microRNA from cancer cells via target-induced cascade amplification generation of lighting-up RNA aptamers

The abnormal expression levels of miRNAs have been proven to be highly related to the generation of various diseases and are also closely associated with the stages and types of disease development. The novel RNA aptamers-based homogenous fluorescent methods were simple, with low background signal and high signal-to-noise ratio, but lacked effective signal amplification technology to achieve sensitive detection of trace miRNA markers. There is an urgent need for combining effective nucleic acid amplification technology with RNA aptamer to achieve highly sensitive and accurate detection of miRNA. For this purpose, a new DNA multi-arm nanostructure-based dual rolling circle transcription machinery for the generation of lighting-up MG RNA aptamers is constructed for label-free and highly sensitive sensing of miRNA-21. In this system, the target miRNA-21 induces a structural transformation of the DNA multi-arm nanostructure probe to recycle miRNA-21 and trigger two independent rolling circle transcription reactions to generate two long RNAs, which can partially hybridize with each other to generate large amounts of complete MG RNA aptamers. These RNA aptamers can associate with organic MG dye to produce significantly enhanced fluorescence signals to accomplish ultrasensitive miRNA-21 detection down to 0.9 fM. In addition, this method exhibits high selectivity to distinguish miRNA-21 even with single nucleotide mismatch, and also has potential application capability to monitor different expression levels of miRNA-21 from different cancer cells. The effective collaboration between MG RNA aptamer and rolling circle transcription reaction makes this fluorescent method show the significant advantages of low background signal, high signal-to-noise ratio and high detection sensitivity. It has great potential to be a promising means to achieve label-free and highly sensitive monitoring of other trace biological markers via a simple change of target sequence.

Development of a one-pot and sequence-specific LAMP assay based on the self-quenching probe integrated by the complementary oligonucleotide for an enhanced fluorescence quenching

Single-modified fluorogenic primer (Sfp) enables accurate identification of LAMP amplicons without being affected by non-specific products. However, the fluorescence self-quenching by nucleobases for Sfp is generally of low efficiency, and the high background signal makes it a great challenge to achieve visual inspection with naked eyes. In the present study, the oligonucleotide (Ao) complementary to Sfp was designed, which would hybridize to Sfp and dramatically heighten the quenching effect, leading to a low background signal in negative reaction. Instead, for positive reaction, Sfp is incorporated into the double-stranded amplicons, resulting in dequenching and consequently, enhanced fluorescence. The detection scheme can be further improved by a dual-color fluorescence strategy, allowing visual detection of 1 pg rainbow trout DNA in a closed-tube format within 30 min. Therefore, our LAMP-Ao-Sfp assay represents a useful tool for rapid and sensitive detection, and can serve as a reliable method for on-site detection in low-resource settings.

Behind the glow: unveiling the nature of NanoLuc reactants and products.

Due to the largely recognized utility of bioluminescence in many fields, a wide variety of luciferase-luciferin systems have been investigated in order to find the best-suited for a number of different applications. The collected knowledge has allowed the identification of a few necessary, or at least desirable, properties, such as bright luminescence, low background signal and small dimension of the enzyme that must exhibit structural stability at operating conditions. The NanoLuc-furimazine pair seems to meet all these requirements, but the mechanism of the reaction and the characteristics of the species responsible for the emission remain unknown. The aim of this study is to identify the luminescent product among the possible forms of oxidized furimazine and to understand how the chemical form and structure of the system, before and after the oxidation, are involved into the reaction mechanism and determine emission. To do this, we consider two possible forms of furimazine, the keto and the enol one, and test which of them is the most plausible candidate in the bioluminescence process on the basis of enzyme-substrate interactions from docking calculations. A similar procedure is repeated for three possible forms of the furimamide luminescent product, and their properties in the protein environment are then evaluated via QM/MM calculations. In contrast with previous indications, our simulations well support the involvement of the enol form of furimazine as reagent and point to the zwitterionic forms of furimamide as emissive species.

A non-peptide-based fluorescent probe capable of sensitively visualizing asparagine endopeptidase.

The non-peptide-based fluorescent probe QMC11 is capable of specifically targeting asparagine endopeptidase (AEP) and imaging cellular endogenous AEP. The motion of the probe can be restricted by AEP to activate fluorescence while keeping a low background signal.

Recent advances in super-resolution optical imaging based on aggregation-induced emission.

Super-resolution imaging has rapidly emerged as an optical microscopy technique, offering advantages of high optical resolution over the past two decades; achieving improved imaging resolution requires significant efforts in developing super-resolution imaging agents characterized by high brightness, high contrast and high sensitivity to fluorescence switching. Apart from technical requirements in optical systems and algorithms, super-resolution imaging relies on fluorescent dyes with special photophysical or photochemical properties. The concept of aggregation-induced emission (AIE) was proposed in 2001, coinciding with unprecedented advancements and innovations in super-resolution imaging technology. AIE probes offer many advantages, including high brightness in the aggregated state, low background signal, a larger Stokes shift, ultra-high photostability, and excellent biocompatibility, making them highly promising for applications in super-resolution imaging. In this review, we summarize the progress in implementation methods and provide insights into the mechanism of AIE-based super-resolution imaging, including fluorescence switching resulting from photochemically-converted aggregation-induced emission, electrostatically controlled aggregation-induced emission and specific binding-regulated aggregation-induced emission. Particularly, the aggregation-induced emission principle has been proposed to achieve spontaneous fluorescence switching, expanding the selection and application scenarios of super-resolution imaging probes. By combining the aggregation-induced emission principle and specific molecular design, we offer some comprehensive insights to facilitate the applications of AIEgens (AIE-active molecules) in super-resolution imaging.

MOF-mediated dual energy transfer nanoprobe integrated with exonuclease III amplification strategy for highly sensitive detection of DNA.

Accurate quantitative detection of DNA is an advanced strategy in various fields (such as disease diagnosis and environmental monitoring), but the classical DNA detection method usually suffers from low sensitivity, expensive thermal cyclers, or strict annealing conditions. Herein, a MOF-ERA platform for ultrasensitive HBV-DNA detection is constructed by integrating metal-organic framework (MOF)-mediated double energy transfer nanoprobe with exonuclease III (Exo III)-assisted target recycling amplification. The proposed double energy transfer containing a donor and two receptors is simply composed of MOFs (UiO-66-NH2, a well-studied MOF) modified with a signal probe formed by the hybridization of carboxyuorescein (FAM)-labeled DNA (FDNA) and black hole quencher (BHQ1)-terminated DNA (QDNA), resulting in low fluorescence signal. After the addition of HBV-DNA, Exo III degradation to FDNA is activated, leading to the liberation of the numerous FAM molecules, followed by the generation of a significant fluorescence signal owing to the negligible binding of MOFs with free FAM molecules. The results certify that the MOF-ERA platform can be successfully used to assay HBV-DNA in the range of 1.0-25.0 nM with a detection limit of 97.2 pM, which is lower than that without BHQ1 or Exo III. The proposed method with the superiorities of low background signal and high selectivity holds promise for early disease diagnosis and clinical biomedicine applications.

Spectro-Electrochemiluminescence Analysis of the Simultaneous Emission of Two Luminophores

Electrochemiluminescence (ECL) is a powerful technique that combines the best features of electrochemistry and chemiluminescence in a single experiment. Its versatility, simplified optical set-up, low background signal and high sensitivity [1] allows the increase of new analytical applications. ECL signals are usually obtained from the excited states of a luminophore generated at the electrode surface during an electrochemical reaction due to the presence of a co-reactant. Particularly, resonance energy transfer (RET) mechanism is produced when at least two luminophores are present in solution, one luminophore acts as donor and the other one as acceptor [2]. In the absence of an external light source, donor luminophore emits light as consequence of its electrochemical excitation, part of this emission is absorbed by the acceptor to be excited and re-emits light.Initially, ECL emission of two luminophores were separately analysed in this work: (1) luminol emission in presence of hydrogen peroxide as co-reactant shows one band from 350 to 600 nm (maximum at 420 nm) during its oxidation process at +0.20 V, while (2) ECL signal of Ru(bpy)3 2+ with tripropylamine as co-reactants displays one band at 620 nm at +0.95 V.Furthermore, the simultaneous emission of luminol and Ru(bpy)3 2 (RET-ECL system) was analysed using a microspectrometer detector [3]. ECL spectra display three emission bands at 420, 510 and 620 nm. According to the previous experiments, bands at 420 and 510 nm should be associated with luminol due to its band emission is located from 350 to 600 nm, while band at 620 nm corresponds to Ru(bpy)3 2+. Nevertheless, Ru(bpy)3 2+ shows a characteristic absorption band at 450 nm, so minimum observed at this wavelength in the spectra is related to the Ru(bpy)3 2+ absorption. It demonstrates that bands at 420 and 510 nm are not really two bands, they form the characteristic band of luminol at 350-600 nm but is separated due to the characteristic absorption of Ru(bpy)3 2+ at 450 nm. Then, it is clear than Ru(bpy)3 2+ absorbs part of ECL emission of luminol before producing their own emission at more positive potentials. Spectro-electrochemiluminescence results demonstrates that the interaction between luminol and Ru(bpy)3 2+ is an important effect to be considered when ECL system is formed by these two luminophores.

The discovery and evaluation of [18F]BMS-986229, a novel macrocyclic peptide PET radioligand for the measurement of PD-L1 expression and in-vivo PD-L1 target engagement.

A same-day PET imaging agent capable of measuring PD-L1 status in tumors is an important tool for optimizing PD-1 and PD-L1 treatments. Herein we describe the discovery and evaluation of a novel, fluorine-18 labeled macrocyclic peptide-based PET ligand for imaging PD-L1. [18F]BMS-986229 was synthesized via copper mediated click-chemistry to yield a PD-L1 PET ligand with picomolar affinity and was tested as an in-vivo tool for assessing PD-L1 expression. Autoradiography showed an 8:1 binding ratio in L2987 (PD-L1 (+)) vs. HT-29 (PD-L1 (-)) tumor tissues, with >90% specific binding. Specific radioligand binding (>90%) was observed in human non-small-cell lung cancer (NSCLC) and cynomolgus monkey spleen tissues. Images of PD-L1 (+) tissues in primates were characterized by high signal-to-noise, with low background signal in non-expressing tissues. PET imaging enabled clear visualization of PD-L1 expression in a murine model in vivo, with 5-fold higher uptake in L2987 (PD-L1 (+)) than in control HT-29 (PD-L1 (-)) tumors. Moreover, this imaging agent was used to measure target engagement of PD-L1 inhibitors (peptide or mAb), in PD-L1 (+) tumors as high as 97%. A novel 18F-labeled macrocyclic peptide radioligand was developed for PET imaging of PD-L1 expressing tissues that demonstrated several advantages within a nonhuman primate model when compared directly to adnectin- or mAb-based ligands. Clinical studies are currently evaluating [18F]BMS-986229 to measure PD-L1 expression in tumors.