Minimal Background Signal Research Articles

Supramolecular Guest Exchange in Cucurbit[7]uril for Bioorthogonal Fluorogenic Imaging across the Visible Spectrum.

Fluorogenic probes that unmask fluorescence signals in response to bioorthogonal reactions are a powerful new addition to biological imaging. They can significantly reduce background fluorescence and minimize nonspecific signals, potentially enabling real-time, high-contrast imaging without the need to wash out excess fluorophores. While diverse classes of highly refined synthetic fluorophores are now readily available, integrating them into a bioorthogonal fluorogenic scheme still requires extensive design efforts and customized structural alterations to optimize quenching mechanisms for each specific fluorophore scaffold. Herein, we present a highly generalizable strategy that can produce an efficient bioorthogonal fluorogenic response from essentially any readily available fluorophore without further structural alterations. We designed this strategy based on the macrocyclic cucurbit[7]uril (CB7) host, where a fluorogenic response is achieved by programming a guest exchange reaction within the macrocyclic cavity. We employed this strategy to rapidly create fluorogenic probes across the visible spectrum from diverse fluorophore scaffolds, which enabled no-wash imaging in live cells and tissues with minimal background signal. Finally, we demonstrated that this strategy can be combined with metabolic labeling for fluorogenic detection of metabolically tagged mycobacteria under no-wash conditions and paired with covalently clickable probes for high-contrast super-resolution and multiplexed imaging in cells and tissues.

A dual-mechanism pretreatment idea for improving the derived Raman spectrum for (micro)plastic analysis

ABSTRACT Raman spectroscopy is an effective tool for microplastic analysis, but its performance can be limited by weak signals and surface interference. This study aims to enhance derived Raman spectra through chemical surface treatments. Fresh and biofouled plastic debris samples, including polyethylene (PE), polypropylene (PP), and polyethylene terephthalate (PET), were treated overnight with various chemical solutions: 30% H2O2, 5% NaOCl, 5% HCl, and a sequential treatment of 5% HCl followed by 5% NaOCl (HCl:NaOCl). Stereo microscope examinations revealed that single-solution treatments removed some stains but left others behind, while complete stain removal was achieved with the HCl:NaOCl treatment. The control samples exhibited a high fluorescence issue, but oxidation pretreatment effectively reduced this, with 5% NaOCl outperforming 30% H2O2. Despite eliminating fluorescence, residual stains still obscured peak signals. In contrast, the sequential HCl:NaOCl treatment removed almost visible stain, resulting in very clean surfaces, minimal background signals, and significantly higher S/N ratios for peak signals. This study demonstrates that dual-mechanism pretreatments – combining acid dissolution and oxidation – are far more effective than single treatments, benefiting applications that require clean plastic samples for subsequent operations.

Hypoxia-responsive synthetic gene circuits to improve safety and potency of CAR T cell therapy for solid tumors.

37 Background: Engineered cell therapies expressing a chimeric antigen receptor (CAR) in human T cells have demonstrated great promise for the treatment of hematological malignancies. However, unique challenges remain in translating the success of CAR T cell therapies to solid tumors, namely: (1) specificity: there are few truly unique antigens present on solid tumors, leading to off-target effects that damage normal tissue and pose safety risks; and (2) suppression: solid tumors develop microenvironments that are immunosuppressive and decrease T cell survival and tumor-directed cytotoxicity. To address these challenges and improve the safety and potency of solid tumor cell therapy, we engineered cells with synthetic genetic circuits containing a previously validated hypoxia biosensor (HBS) that responds to upregulation of the native HIF1α and HIF2α transcription factors by hypoxia, enabling the cells to sense and respond to the hypoxic tumor microenvironment ubiquitous among solid tumors. Methods: We evaluated CAR expression from circuit architectures containing the HBS promoter driving expression of both CAR and native HIF1α, HIF2α, as well as from circuit architectures containing the HBS promoter and our toolkit of synthetic transcription factors and promoters. Circuits were integrated into HEK293 cells via PiggyBac transposon vector to generate stable cell lines. Cells were subsequently cultured in normoxia (21% O2) and hypoxia (1% O2), and harvested for flow cytometric analysis to evaluate CAR surface expression across a three day time frame. Results: We identified novel circuits providing fast reporter output with minimal background signal under hypoxic conditions, and elucidated design rules that enable or restrict amplification of transgene in native HIF1α and HIF2α-containing circuits. We observed differences in CAR transgene expression in hypoxia between circuits containing HIF1α and HIF2α-mediated feedback, suggesting mechanistic differences in regulation of HIF1α and HIF2α when overexpressed. Additionally, we observed greatly amplified transgene expression in circuit topologies containing feedback mediated by our synthetic transcription factor and promoter toolkit compared to native HIF1α and HIF2α-containing circuits and cells constitutively expressing CAR. Conclusions: We present a new therapeutic technology that senses hypoxic environments and enables high-output sense and respond behaviors with therapeutic outputs. We envision that this technology will enable development of safer, more effective cell therapies for solid tumors, as well as provide new biological and mechanistic insights that can inform the future development of hypoxia-responsive cell therapies.

Integrating CdIn2S4 semiconductors with S-vacancy MoS2 nanosheets to fabricate a multi-channel aptasensing chip for photoelectrochemical detection of multiple mycotoxins

BackgroundThe importance of multi-target simultaneous detection lies in its ability to significantly boost detection efficiency, making it invaluable for rapid and cost-effective testing. Photoelectrochemical (PEC) sensors have emerged as promising candidates for detecting harmful substances and biomarkers, attributable to their unparalleled sensitivity, minimal background signal, cost-effectiveness, equipment simplicity, and outstanding repeatability. However, designing an effective multi-target detection strategy remains a challenging task in the PEC sensing field. Consequently, there is a pressing need to address the development of PEC sensors capable of simultaneously detecting multiple targets. ResultsCdIn2S4/V–MoS2 heterojunctions were successfully prepared via a hydrothermal method. These heterojunctions exhibited a high photocurrent intensity, representing a 1.53-fold enhancement compared to CdIn2S4 alone. Next, we designed a multi-channel aptasensing chip using ITO as the substrate. Three working electrodes were created via laser etching and subsequently modified with CdIn2S4/V–MoS2 heterojunctions. Thiolated aptamers were then self-assembled onto the CdIn2S4/V–MoS2 heterojunctions via covalent bonds, serving as recognition tool. By empolying the CdIn2S4/V–MoS2 heterojunctions as the sensing platform and aptamers as recognition tool, we successfully developed a disposable aptasensing chip for the simultaneous PEC detection of three typical mycotoxins (aflatoxin B1 (AFB1), ochratoxin A (OTA), and zearalenone (ZEN)). This aptasensing chip exhibited wide detection range for AFB1 (0.05–50 ng/mL), OTA (0.05–500 ng/mL), and ZEN (0.1–250 ng/mL). Furthermore, it demonstrated ultra-low detection limits of 0.017 ng/mL for AFB1, 0.016 ng/mL for OTA, and 0.033 ng/mL for ZEN. Significance and noveltyThe aptasensing chip stands out for its cost-effectiveness, simplicity of fabrication, and multi-channel capabilities. The versatility and practicality enable it to serve as a powerful platform for designing multi-channel PEC aptasensors. With its ability to detect multiple targets with high sensitivity and specificity, the aptasensing chip holds immense potential for applications across diverse fields, such as environmental monitoring, clinical diagnostics, and food safety monitoring, where multi-target detection is crucial.

Exonuclease-iii -propelled DNAzyme cascade for sensitive and reliable cervical cancer related miRNA analysis

MicroRNAs (miRNAs) can serve as biomarkers for early-diagnosis, therapy, and postoperative care of cervical cancer. Sensitive and reliable quantification of miRNA remains a huge challenge due to its low expressing levels and background interference. Herein, we propose a novel exonuclease-III (Exo–III)–propelled DNAzyme cascade for sensitive and high-efficient miRNA analysis. This method involves the engineering of compact DNAzyme hairpin probes, including the H1 probe and H2 probe. The H1 probe is designed with exposed analyte recognition subunits that can specifically recognize target miRNA. This recognition triggers two processes: Exo-iii-assisted target regeneration and successive substrate cleavage catalyzed by DNAzyme. The unique character of Exo-III that catalyzes removal of mononucleotides from the blunt or recessed 3′-OH termini of dsDNA confers the approach with a minimal background signal. The multiple signal cycles provided an abundant signal amplification and consequently, the method exhibited a low limit of detection of 3.12 fM, and a better specificity over several homologous miRNAs. In summary, this powerful Exo-III driven DNAzyme cascaded system offers broader and more adaptable methods for comprehending the activities of miRNA in various biological occurrences.

Unmasking the Electrochemiluminescence Properties of Ternary Mn/Fe/Co Metals Doped Porous g-C3N4 Fiber-like Nanostructure.

Graphitic-phase carbon nitride (g-C3N4) materials have exhibited increasingly remarkable performance as emerging electrochemiluminescence (ECL) emitters, owing to their unique optical and electronic properties; however, the ECL merits of porous g-C3N4 nanofibers doped with ternary metals are not yet explored. Deciphering the ECL properties of trimetal-doped g-C3N4 nanofibers could provide an exquisite pathway for ultrasensitive sensing and imaging with impressive advantages of minimal background signal, great sensitivity, and durability. Herein, we rationally synthesized g-C3N4 nanofibers doped atomically with Mn, Fe, and Co elements (Mn/Fe/Co/g-C3N4) in a one-pot via the protonation in ethanol and annealing process driven by the rolling up mechanism. The ECL performance of g-C3N4 with and without metal dopants was investigated and compared with standard Ru(bpy)32+ in the presence of potassium persulfate (K2S2O8) as the coreactant. Notably, g-C3N4 nanofibers doped with metal ions exhibited an ECL efficiency of 483% that was 4.83 times higher than that of Ru(bpy)32+. Mechanistic investigations unveiled that the g-C3N4 nanofibers possess a large surface area and, as a result, exhibit a reduced interfacial impedance within the porous microstructure. These factors contribute to the acceleration of charge transfer rates and the stabilization of charge carriers and excitons, ultimately facilitating the ECL process. This research endeavor may pave the way for a new hot research area and serves as a powerful tool for elucidating fundamental inquiries of ECL on one-dimensional g-C3N4 nanostructures.

Fluorescein-switching-based lateral flow assay for the detection of microRNAs.

Lateral flow assays (LFAs) are a cost-effective and rapid colorimetric technology that can be effectively used for nucleic acid tests (NATs) in various fields such as medical diagnostics and biotechnology. Given their importance, developing more diverse LFAs that operate through novel working mechanisms is essential for designing highly selective and sensitive NATs and providing insights for designing various practical point-of-care testing (POCT) systems. Herein we report a new type of lateral flow assay (LFA) based on fluorescein-switching, enabled by nucleic acid-templated photooxidation of reduced fluorescein by riboflavin tetraacetate (RFTA). The LFA design leverages the fact that a reduced form of fluorescein, which weakly binds to gold nanoparticle (GNP)-conjugated anti-fluorescein antibodies, is oxidized in the presence of target nucleic acids to yield its native state, which then strongly binds to the antibodies. The study involved designing and optimizing probe sequences to detect miR-6090 and miR-141, which are significant markers for prostate cancer. To minimize background signals of LFAs, sodium borohydride (NaBH4) was specifically introduced as a reducing agent, and detailed procedures were established. The developed LFA system accurately identified low fmol levels of target microRNAs with minimal false positives, all detectable with the naked eye, making the system a promising tool for point-of-care diagnostics.

Near-Infrared Optogenetic Module for Conditional Protein Splicing

Optogenetics has emerged as a powerful tool for spatiotemporal control of biological processes. Near-infrared (NIR) light, with its low phototoxicity and deep tissue penetration, holds particular promise. However, the optogenetic control of polypeptide bond formation has not yet been developed. In this study, we introduce a NIR optogenetic module for conditional protein splicing (CPS) based on the gp41-1 intein. We optimized the module to minimize background signals in the darkness and to maximize the contrast between light and dark conditions. Next, we engineered a NIR CPS gene expression system based on the protein ligation of a transcription factor. We applied the NIR CPS for light-triggered protein cleavage to activate gasdermin D, a pore-forming protein that induces pyroptotic cell death. Our NIR CPS optogenetic module represents a promising tool for controlling molecular processes through covalent protein linkage and cleavage.

A red-emitting ultrasensitive fluorescent probe for specific detection and biological visualization of cysteine in vitro and in vivo

Developing efficient strategies for specific detection of cysteine (Cys) is of great importance for identifying complicated biological roles in physiological and pathological processes. Herein, an ultrasensitive red-emission fluorescent probe (termed 1) is constructed for specific detection and biological visualization of Cys. The linked-anthocyanin fluorophore modified with a twisted N, N-diethylamino moiety shows improved red-shifted emission (642 nm) and absolute quantum yield (0.224 in dimethyl sulfoxide), as well as minimal fluorescence background signal and good water solubility. Meanwhile, utilizing acryloyl chloride as recognition group endows the probe 1 with excellent sensitivity and selectivity towards Cys (limit of detection: 2.93 nM). More importantly, the in vitro and in vivo results confirm that the probe 1 has the capacity of fluorescence imaging of Cys and good biological safety, which holds great promise for bioanalysis and biosensing of Cys.

Emerging Microfluidic Devices for Sample Preparation of Undiluted Whole Blood to Enable the Detection of Biomarkers.

Blood testing allows for diagnosis and monitoring of numerous conditions and illnesses; it forms an essential pillar of the health industry that continues to grow in market value. Due to the complex physical and biological nature of blood, samples must be carefully collected and prepared to obtain accurate and reliable analysis results with minimal background signal. Examples of common sample preparation steps include dilutions, plasma separation, cell lysis, and nucleic acid extraction and isolation, which are time-consuming and can introduce risks of sample cross-contamination or pathogen exposure to laboratory staff. Moreover, the reagents and equipment needed can be costly and difficult to obtain in point-of-care or resource-limited settings. Microfluidic devices can perform sample preparation steps in a simpler, faster, and more affordable manner. Devices can be carried to areas that are difficult to access or that do not have the resources necessary. Although many microfluidic devices have been developed in the last 5 years, few were designed for the use of undiluted whole blood as a starting point, which eliminates the need for blood dilution and minimizes blood sample preparation. This review will first provide a short summary on blood properties and blood samples typically used for analysis, before delving into innovative advances in microfluidic devices over the last 5 years that address the hurdles of blood sample preparation. The devices will be categorized by application and the type of blood sample used. The final section focuses on devices for the detection of intracellular nucleic acids, because these require more extensive sample preparation steps, and the challenges involved in adapting this technology and potential improvements are discussed.

Engineering a modular double-transmembrane synthetic receptor system for customizing cellular programs

Implementation of designer receptors in engineered cells confers them to sense a particular physiological or disease state and respond with user-defined programs. To expand the therapeutic application scope of engineered cells, synthetic receptors realized through different strategies are in great demand. Here, we develop a synthetic receptor system that exerts dual control by incorporating two transmembrane helices for the signal chain. Together with a sensor-actuator device with minimal background signals and a positive loop circuit, this receptor system can sensitively respond to extracellular protein signals. We demonstrate that this synthetic receptor system can be readily adapted to respond to various inputs, such as interleukin-1 (IL-1), programmed death ligand 1 (PD-L1), and HER2, and release customized outputs, including fluorescence signals and the therapeutic molecule IL-2. The robust signaling ability and generality of this receptor system promise it to be a useful tool in the field of cell engineering for fundamental research and translational applications.

Three-dimensional feature matching improves coverage for single-cell proteomics based on ion mobility filtering.

SUMMARYSingle-cell proteomics (scProteomics) promises to advance our understanding of cell functions within complex biological systems. However, a major challenge of current methods is their inability to identify and provide accurate quantitative information for low-abundance proteins. Herein, we describe an ion-mobility-enhanced mass spectrometry acquisition and peptide identification method, transferring identification based on FAIMS filtering (TIFF), to improve the sensitivity and accuracy of label-free scProteomics. TIFF extends the ion accumulation times for peptide ions by filtering out singly charged ions. The peptide identities are assigned by a three-dimensional MS1 feature matching approach (retention time, accurate mass, and FAIMS compensation voltage). The TIFF method enabled unbiased proteome analysis to a depth of >1,700 proteins in single HeLa cells, with >1,100 proteins consistently identified. As a demonstration, we applied the TIFF method to obtain temporal proteome profiles of >150 single murine macrophage cells during lipopolysaccharide stimulation and identified time-dependent proteome changes. A record of this paper’s transparent peer review process is included in the supplemental information.

Direct imaging of single-molecule electrochemical reactions in solution.

Chemical reactions tend to be conceptualized in terms of individual molecules transforming into products, but are usually observed in experiments that probe the average behaviour of the ensemble. Single-molecule methods move beyond ensemble averages and reveal the statistical distribution of reaction positions, pathways and dynamics1-3. This has been shown with optical traps and scanning probe microscopy manipulating and observing individual reactions at defined locations with high spatial resolution4,5, and with modern optical methods using ultrasensitive photodetectors3,6,7 that enable high-throughput single-molecule measurements. However, effective probing of single-molecule solution chemistry remains challenging. Here we demonstrate optical imaging of single-molecule electrochemical reactions7 in aqueous solution and its use for super-resolution microscopy. The method utilizes a chemiluminescent reaction involving a ruthenium complex electrochemically generated at an electrode8, which ensures minimal background signal. This allows us to directly capture single photons of the electrochemiluminescence of individual reactions, and to develop super-resolved electrochemiluminescence microscopy for imaging the adhesion dynamics of live cells with high spatiotemporal resolution. We anticipate that our method will advance the fundamental understanding of electrochemical reactions and prove useful for bioassays and cell-imaging applications.

Abstract 1676: A comprehensive whole blood CyTOF immune monitoring panel with expanded surface and intracellular markers using the Maxpar Direct Immune Profiling Assay

Abstract In-depth monitoring of the immune response to cancer and infection is vital to ascertain disease status and to assess immunotherapeutic options. Time-of-flight technology, the basis of CyTOF® mass cytometry, enables multiplex proteomic cellular phenotyping with 50 or more markers, making it ideal for comprehensive immune profiling. Unlike fluorescence-dependent approaches, which require signal compensation that makes the development of larger panels more challenging, CyTOF utilizes monoisotopic metal-tagged antibodies that exhibit minimal background signal, enabling the highest-resolution multiparametric landscape of a single cell. The Maxpar® Direct™ Immune Profiling Assay™ is a pre-titrated dried-down 30-antibody cocktail preparation for immune profiling of human whole blood or PBMC. This assay is used with Maxpar Pathsetter™ software, which resolves whole blood into 37 immune populations comprising major lineage populations and their subsets, such as CD4 Th subsets and B and T cell memory cells, as well as stratifications of myeloid populations. The resulting system is a simple sample-to-answer solution for immune monitoring studies. Here, we expanded the 30-marker assay with 14 additional antibodies comprising pertinent targets of immunotherapy, including the exhaustion markers PD-1, PD-L1, Tim-3 and CTLA-4, and co-stimulation markers 4-1BB and ICOS. We also demonstrated the compatibility of the assay with downstream intracellular staining for cytoplasmic markers IFN-γ, TNF-α, IL-2, perforin and granzyme B for assessment of cellular function in 4h PMA/ionomycin-stimulated whole blood cultures. Next, we modified the existing Pathsetter model to automate the analysis of whole blood stained with the expanded panel to generate reports on key immune cell populations, percentages of exhausted cells and cell subsets producing cytokines. Last, to demonstrate the ability of this expanded panel to identify antigen-specific T cell subsets accompanied by their in-depth phenotypic assessment, we tested this panel against CMV peptide-stimulated whole blood samples. We demonstrated the flexibility of the Maxpar Direct Immune Profiling Assay in panel customization and a streamlined workflow for automated analysis to enable comprehensive immune profiling of human whole blood. For Research Use Only. Not for use in diagnostic procedures. Citation Format: Shariq Mujib, Stephen K. Li, Nick Zabinyakov, Christina Loh. A comprehensive whole blood CyTOF immune monitoring panel with expanded surface and intracellular markers using the Maxpar Direct Immune Profiling Assay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1676.

TZM-gfp cells: a tractable fluorescent tool for analysis of rare and early HIV-1 infection

Here we describe TZM-gfp, a novel HIV-1 reporter cell derived from the same parental clone JC.53, used previously to generate the widely-utilized indicator cell line TZM-bl. We re-engineered JC.53 cells to express GFP under regulation of HIV Tat and Rev. We characterize the new reporter cell line to show that TZM-gfp cells are equally susceptible to HIV infection, exhibit minimal background signal, and can report HIV infection in rare cells from a bulk population of experimentally-infected human monocyte-derived macrophages. We demonstrate the utility and sensitivity of the cells in detection of even a single HIV-positive macrophage by fluorescence-assisted correlative electron microscopy, using the GFP signal to guide imaging of HIV virions in primary co-culture. Finally, we used TZM-gfp cells for viral capture during co-culture with human peripheral blood mononuclear cells, showing that TZM-gfp can support outgrowth and analyses of patient-derived primary HIV-1 isolates.

Quantification of CRP in human serum using a handheld fluorescence detection system for capillary-based ELISA

We developed a handheld fluorescence detection system for capillary-based enzyme-linked immunosorbent assay (ELISA). The detection system implements both a long-pass filter and perpendicular optical arrangement, i.e., a power LED and a palm-sized spectrometer, to minimize background signals from the excitation light and optical scattering. The lower detection limit for resorufin was 0.13 μM. The detection system was applied to the quantification of C-reactive protein (CRP) in human serum with a capillary-based ELISA. The lower detection limit for CRP was 31 ng/ml, and the observed CRP levels in human serum were comparable to those obtained with a conventional ELISA system.

Mass spectrometric analyses of high performance polymers to assess their radiopurity as ultra low background materials for rare event physics detectors

The mass concentrations of 232Th and 238U in several strong, unfilled, high performance polymers are reported as measures of their radiopurity. Highly radiopure polymers are required as dielectric materials in the construction of rare event physics detectors, in order to minimize background signals arising from the detector materials themselves. New data are reported for carefully sourced samples of polyetheretherketone (PEEK), polyetherketoneketone (PEKK), a polyamide imide (PAI, branded as Torlon) and polybenzimidazole (PBI). Data for solid polyetherimide (PEI) are also discussed and new data for PEI in the form of a commercial 3D printing filament stock are reported. Strong high performance polymers PEKK, PBI, PAI and PEI were found with levels for 232Th and 238U that are below one mBq/kg, including the PEI 3D printing filament. Specifically, for 232Th and 238U respectively, in μBq/kg (emphasis “micro”Bq/kg), we found values of 149 and 184 for Arkema Kepstan PEKK 6002 flake; 69 and 2250 for Solvay Ketaspire PEEK flake; 346 and 291 for PBI Performance Products low metals grade 100 mesh PBI powder; 66 and 105 for Drake Plastics PAI Torlon T-4200 pellets; 401 and 285 for Drake Plastics cured PAI rod; 32 and 41 for Ensinger PEI Ultem 1000 solid; and 15 and 85 μBq/kg, for ThermaX PEI Ultem 1010 filament material. These results were all obtained using a novel dry ashing method in crucibles constructed of ultra low background (ULB) electroformed copper. Samples were spiked with 229Th and 233U as internal standards prior to ashing and determinations were made by inductively coupled plasma mass spectrometry (ICP-MS). Radiopurity is displayed graphically relative to numerical measures of mechanical strength for these and several other polymers.

129Xe NMR-Protein Sensor Reveals Cellular Ribose Concentration.

Dysregulation of cellular ribose uptake can be indicative of metabolic abnormalities or tumorigenesis. However, analytical methods are currently limited for quantifying ribose concentration in complex biological samples. Here, we utilize the highly specific recognition of ribose by ribose-binding protein (RBP) to develop a single-protein ribose sensor detectable via a sensitive NMR technique known as hyperpolarized 129Xe chemical exchange saturation transfer (hyper-CEST). We demonstrate that RBP, with a tunable ribose-binding site and further engineered to bind xenon, enables the quantitation of ribose over a wide concentration range (nM to mM). Ribose binding induces the RBP "closed" conformation, which slows Xe exchange to a rate detectable by hyper-CEST. Such detection is remarkably specific for ribose, with the minimal background signal from endogenous sugars of similar size and structure, for example, glucose or ribose-6-phosphate. Ribose concentration was measured for mammalian cell lysate and serum, which led to estimates of low-mM ribose in a HeLa cell line. This highlights the potential for using genetically encoded periplasmic binding proteins such as RBP to measure metabolites in different biological fluids, tissues, and physiologic states.

Two-way magnetic resonance tuning and enhanced subtraction imaging for non-invasive and quantitative biological imaging.

Distance-dependent magnetic resonance tuning (MRET) technology enables the sensing and quantitative imaging of biological targets in vivo, with the advantage of deep tissue penetration and less interactions with the surroundings as compared to fluorescence-based Förster resonance energy transfer (FRET). However, applications of MRET technology in vivo are currently limited by the moderate contrast enhancement and stability of T1-based MRET probes. Here we report a new two-way magnetic resonance tuning (t-MRET) nanoprobe with dually activatable T1 and T2 magnetic resonance signals that is coupled with dual-contrast enhanced subtraction imaging (DESI). This integrated platform achieves substantially improved contrast enhancement with minimal background signal and can be used to quantitatively image molecular targets in tumours and to sensitively detect very small intracranial brain tumours in patient-derived xenograft models. The high tumour-to-normal tissue ratio offered by t-MRET in combination with DESI provides new opportunities for molecular diagnostics and image-guided biomedical applications.

Improved sensitivity for ratiometric fluorescence detection of ricin based on “kinetic competition” aptasensing strategy

The aptamer recognition mechanisms have been well developed based on equilibration between aptamer conformations modulated by ligand, and can be compatible with almost all kinds of readout techniques for aptamer-based biosensors (i.e., aptasensors). A novel and robust aptasensing strategy named “kinetic competition” has also been investigated in which the blocker kinetically competes the aptamer and probe DNA (e.g., molecular beacon). It improved general methodology for developing aptasensors because it does not require a lengthy pre-equilibration, but the pre-binding of target and its aptamer alters kinetic competition for transducing signal. However, some disadvantages still limited its practical applications, such as high background signal, low sensitivity and accuracy in complex matrices and so on. Herein, we firstly reported the ratiometric “kinetic competition” aptasensor for ricin A chain (RTA) detection. It can well avoid false positive results and minimize background signals, which achieves more sensitive, accurate, reliable and effective detection. Under optimal conditions, this strategy effectively improved the sensitivity and reduced the background signal. A limit of detection (LOD) of 0.23 nM for RTA could be achieved without any separation and amplification techniques. The recovery data (73.8 %–111.0 %) was also satisfactory for RTA analysis in food powder samples and serum matrix.