Affibody Functionalized Beads for the Highly Sensitive Detection of Cancer Cell-Derived Exosomes.

Exosomes belong to the class of extracellular vesicles of endocytic origin, which are regarded as a promising source of cancer biomarkers in liquid biopsy. As a result, an accurate, sensitive, and specific quantification of these nano-sized particles is of significant importance. Affinity-based approaches are recognized as the most valuable technique for exosome isolation and characterization. Indeed, Affibody biomolecules are a type of protein scaffold engineered with small size and enjoy the features of high thermal stability, affinity, and specificity. While the utilization of antibodies, aptamers, and other biologically active substances for exosome detection has been reported widely, there are no reports describing Affibody molecules' usage for exosome detection. In this study, for the first time, we have proposed a novel strategy of using Affibody functionalized microbeads (AffiBeads) for exosome detection with a high degree of efficiency. As a proof-of-concept, anti-EGFR-AffiBeads were fabricated and applied to capture and detect human lung A549 cancer cell-derived EGFR-positive exosomes using flow cytometry and fluorescent microscopy. Moreover, the capture efficiency of the AffiBeads were compared with its counterpart antibody. Our results showed that the Affibody probe had a detection limit of 15.6 ng exosomes per mL (~12 exosomes per AffiBead). The approach proposed in the current study can be used for sensitive detection of low expression level markers on tumor-derived exosomes, providing a basis for early-stage cancer diagnosis.

A Robust Intrinsically Green Fluorescent Poly(Amidoamine) Dendrimer for Imaging and Traceable Central Nervous System Delivery in Zebrafish.

Intrinsically fluorescent poly(amidoamine) dendrimers (IF-PAMAM) are an emerging class of versatile nanoplatforms for in vitro tracking and bio-imaging. However, limited tissue penetration of their fluorescence and interference due to auto-fluorescence arising from biological tissues limit its application in vivo. Herein, a green IF-PAMAM (FGP) dendrimer is reported and its biocompatibility, circulation, biodistribution and potential role for traceable central nervous system (CNS)-targeted delivery in zebrafish is evaluated, exploring various routes of administration. Key features of FGP include visible light excitation (488 nm), high fluorescence signal intensity, superior photostability and low interference from tissue auto-fluorescence. After intravenous injection, FGP shows excellent imaging and tracking performance in zebrafish. Further conjugating FGP with transferrin (FGP-Tf) significantly increases its penetration through the blood-brain barrier (BBB) and prolongs its circulation in the blood stream. When administering through local intratissue microinjection, including intracranial and intrathecal injection in zebrafish, both FGP and FGP-Tf exhibit excellent tissue diffusion and effective cellular uptake in the brain and spinal cord, respectively. This makes FGP/FGP-Tf attractive for in vivo tracing when transporting to the CNS is desired. The work addresses some of the major shortcomings in IF-PAMAM and provides a promising application of these probes in the development of drug delivery in the CNS.

Visualizing neuroinflammation with fluorescence and luminescent lanthanide-based in situ hybridization.

BACKGROUND: Neurokine signaling via the release of neurally active cytokines arises from glial reactivity and is mechanistically implicated in central nervous system (CNS) pathologies such as chronic pain, trauma, neurodegenerative diseases, and complex psychiatric illnesses. Despite significant advancements in the methodologies used to conjugate, incorporate, and visualize fluorescent molecules, imaging of rare yet high potency events within the CNS is restricted by the low signal to noise ratio experienced within the CNS. The brain and spinal cord have high cellular autofluorescence, making the imaging of critical neurokine signaling and permissive transcriptional cellular events unreliable and difficult in many cases. METHODS: In this manuscript, we developed a method for background-free imaging of the transcriptional events that precede neurokine signaling using targeted mRNA transcripts labeled with luminescent lanthanide chelates and imaged via time-gated microscopy. To provide examples of the usefulness this method can offer to the field, the mRNA expression of toll-like receptor 4 (TLR4) was visualized with traditional fluorescent in situ hybridization (FISH) or luminescent lanthanide chelate-based in situ hybridization (LISH) in mouse BV2 microglia or J774 macrophage phenotype cells following lipopolysaccharide stimulation. TLR4 mRNA staining using LISH- and FISH-based methods was also visualized in fixed spinal cord tissues from BALB/c mice with a chronic constriction model of neuropathic pain or a surgical sham model in order to demonstrate the application of this new methodology in CNS tissue samples. RESULTS: Significant increases in TLR4 mRNA expression and autofluorescence were visualized over time in mouse BV2 microglia or mouse J774 macrophage phenotype cells following lipopolysaccharide (LPS) stimulation. When imaged in a background-free environment with LISH-based detection and time-gated microscopy, increased TLR4 mRNA was observed in BV2 microglia cells 4 h following LPS stimulation, which returned to near baseline levels by 24 h. Background-free imaging of mouse spinal cord tissues with LISH-based detection and time-gated microscopy demonstrated a high degree of regional TLR4 mRNA expression in BALB/c mice with a chronic constriction model of neuropathic pain compared to the surgical sham model. CONCLUSIONS: Advantages offered by adopting this novel methodology for visualizing neurokine signaling with time-gated microscopy compared to traditional fluorescent microscopy are provided.

Chemoenzymatic glycan labelling as a platform for site-specific IgM-antibody drug conjugates.

Immunoglobulin M (IgM) type antibodies play a significant role in complement activation, cellular debris clearance and cell quality control, and have the potential to be used as a therapeutic or targeting/delivery antibody. However, this potential has not been explored thoroughly due to its high molecular weight, polymeric structure and large number of glycosylation sites. Site-specific antibody-drug-conjugates (ADC) are considered the next generation protein biotherapeutic drugs and currently all, in clinical trials and approved, are of the IgG isotype. As existing methods for the development and characterization of IgG-ADCs are not compatible with IgM-ADC, we describe a platform methodology suitable for site specific IgM-ADC using a chemoenzymatic method targeting the glycans on the IgM. Azide functionalized sialic acids were incorporated onto IgM glycans using sialyltransferase for biocompatible conjugation using "click" chemistry. The number of azide groups incorporated onto the IgM glycans were characterized by mass spectrometry of the enzymatically released glycans and glycopeptides. Quantitation of the azide incorporation showed an azide antibody ratio of 8 (glycan data) and 6-10 (glycopeptide data) which translates to a high drug antibody ratio based on IgG-ADC standards. This platform methodology can be readily adapted for any human IgM produced in a mammalian cell expression system.

Time-Gated Luminescent In Situ Hybridization (LISH): Highly Sensitive Detection of Pathogenic Staphylococcus aureus.

We describe simple direct conjugation of a single TEGylated Europium chelate to DNA that binds to intracellular rRNA and is then detected using a homogeneous luminescent in situ hybridisation (LISH) technique. As a proof-of-principle, Staphylococcus aureus (S. aureus) was selected as a model for our study to show the ability of this probe to bind to intracellular 16S ribosomal rRNA. A highly purified Europium chelate conjugated oligonucleotide probe complementary to an rRNA sequence-specific S. aureus was prepared and found to be soluble and stable in aqueous solution. The probe was able to bind specifically to S. aureus via in situ hybridisation to differentiate S. aureus from a closely related but less pathogenic Staphylococcus species (S. epidermidis). A time-gated luminescent (TGL) microscope system was used to generate the high signal-to-noise ratio (SNR) images of the S. aureus. After excitation (365 nm, Chelate λmax = 335 nm), the long-lived (Eu3+) luminescent emission from the probe was detected without interference from natural background autofluorescence typically seen in biological samples. The luminescent images were found to have 6 times higher SNR or sensitivity compared to the fluorescent images using conventional fluorophore Alexa Fluor 488. The TEGylated Europium chelate -oligo probe stained S. aureus with mean signal intensity 3.5 times higher than the threshold level of signal from S. epidermidis (with SNR 8 times higher). A positive control probe (EUB338-BHHTEGST-Eu3+) has mean signal intensity for S. aureus and S. epidermidis equally 3.2 times higher than the threshold of signal for a negative NON-EUB338 control probe. The direct conjugation of a single Europium chelate to DNA provides simplicity and improvement over existing bovine serum albumin (BSA)/streptavidin/biotinylated DNA platforms for multi-attachment of Europium chelate per DNA and more importantly makes it feasible for hybridisation to intracellular RNA targets. This probe has great potential for highly sensitive homogeneous in situ hybridisation detection of the vast range of intracellular DNA targets.

Sensitive Time-Gated Immunoluminescence Detection of Prostate Cancer Cells Using a TEGylated Europium Ligand.

We describe the application of a synthetically developed tetradentate ß-diketonate-europium chelate with high quantum yield (39%), for sensitive immunodetection of prostate cancer cells (DU145). MIL38 antibody, a mouse monoclonal antibody against Glypican 1, conjugated directly to the chelate via lysine residues, resulted in soluble (hydrophilic) and stable immunoconjugates. Indirect labeling of the antibody by a europium chelated secondary polyclonal antibody and a streptavidin/biotin pair was also performed. All of these bright luminescent conjugates were used to stain DU145 cells, a prostate cancer cell line, using time gated luminescence microscopy for imaging, and their performances were compared to conventional FITC labeling. For all prepared conjugates, the europium chelate in conjunction with a gated autosynchronous luminescence detector (GALD) completely suppressed the cellular autofluorescence background to allow capture of vivid, high contrast images of immune-stained cancer cells.

Synthesis and photophysical investigations of pyridine-pyrazolate bound boron(III) diaryl complexes.

This study presents the design and synthetic pathway of unsymmetric ligands based on pyridine-pyrazolate scaffold with Donor-Acceptor (D-A) molecular arrays and their boron complexes to achieve a large Stokes shift. Intermolecular charge transfer (ICT) triggered by the uneven molecular charge distribution from electronically dense pyrazolate (donor) part of the ligands to electron-deficient boron centre (acceptor) resulted in a mega Stokes shift up to 263 nm for selected compounds while retaining the characteristic quantum efficiency and chemical stability. The photophysical properties of derivatization of pyrazolate group in the pyridine-pyrazolate scaffold of diaryl boron complexes were explored based on UV-Visible, steady-state and time-resolved fluorescence spectroscopy. An interesting dual emission along with quenching behaviour was also observed for 2-(6-methoxynaphthelene) 5-(2-pyridyl) pyrazolate boron complex (P5) due to the formation of a twisted intermolecular charge transfer (TICT) state from a locally excited (LE) state rendering it a potential candidate for sensing applications based on H-Bond quenching. In addition, the extended excited state lifetime of the reported compounds compared to classical boron-dipyrromethene (BODIPY) makes them suitable as potential probes for analytical applications requiring a longer excited state lifetime.

Detection of rare prostate cancer cells in human urine offers prospect of non-invasive diagnosis.

Two molecular cytology approaches, (i) time-gated immunoluminescence assay (TGiA) and (ii) Raman-active immunolabeling assay (RiA), have been developed to detect prostate cancer (PCa) cells in urine from five prostate cancer patients. For TGiA, PCa cells stained by a biocompatible europium chelate antibody-conjugated probe were quantitated by automated time-gated microscopy (OSAM). For RiA, PCa cells labeled by antibody-conjugated Raman probe were detected by Raman spectrometer. TGiA and RiA were first optimized by the detection of PCa cultured cells (DU145) spiked into control urine, with TGiA-OSAM showing single-cell PCa detection sensitivity, while RiA had a limit of detection of 4-10 cells/mL. Blinded analysis of each patient urine sample, using MIL-38 antibody specific for PCa cells, was performed using both assays in parallel with control urine. Both assays detected very low abundance PCa cells in patient urine (3-20 PCa cells per mL by TGiA, 4-13 cells/mL by RiA). The normalized mean of the detected PCa cells per 1 ml of urine was plotted against the clinical data including prostate specific antigen (PSA) level and Clinical Risk Assessment for each patient. Both cell detection assays showed correlation with PSA in the high risk patients but aligned with the Clinical Assessment rather than with PSA levels of the low/intermediate risk patients. Despite the limited available urine samples of PCa patients, the data presented in this proof-of-principle work is promising for the development of highly sensitive diagnostic urine tests for PCa.

Point of Care Diagnostics in the Age of COVID-19.

The recent outbreak of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its associated serious respiratory disease, coronavirus disease 2019 (COVID-19), poses a major threat to global public health. Owing to the lack of vaccine and effective treatments, many countries have been overwhelmed with an exponential spread of the virus and surge in the number of confirmed COVID-19 cases. Current standard diagnostic methods are inadequate for widespread testing as they suffer from prolonged turn-around times (>12 h) and mostly rely on high-biosafety-level laboratories and well-trained technicians. Point-of-care (POC) tests have the potential to vastly improve healthcare in several ways, ranging from enabling earlier detection and easier monitoring of disease to reaching remote populations. In recent years, the field of POC diagnostics has improved markedly with the advent of micro- and nanotechnologies. Due to the COVID-19 pandemic, POC technologies have been rapidly innovated to address key limitations faced in existing standard diagnostic methods. This review summarizes and compares the latest available POC immunoassay, nucleic acid-based and clustered regularly interspaced short palindromic repeats- (CRISPR)-mediated tests for SARS-CoV-2 detection that we anticipate aiding healthcare facilities to control virus infection and prevent subsequent spread.

Lipopolysaccharide and Morphine-3-Glucuronide-Induced Immune Signalling Increases the Expression of Polysialic Acid in PC12 Cells.

Polysialic acid (polySia), a long homopolymer of 2,8-linked sialic acids, is abundant in the embryonic brain and is restricted largely in adult brain to regions that exhibit neurogenesis and structural plasticity. In the central nervous system (CNS), polySia is highly important for cell-cell interactions, differentiation, migration and cytokine responses, which are critical neuronal functions regulating intercellular interactions that underlie immune signalling in the CNS. In recent reports, a metabolite of morphine, morphine-3-glucuronide (M3G), has been shown to cause immune signalling in the CNS. In this study, we compared the effects of neurite growth factor (NGF), lipopolysaccharide (LPS) and M3G exposure on the expression of polySia in PC12 cells using immunocytochemistry and Western blot analysis. PolySia was also extracted from stimulated cell proteins by endo-neuraminidase digestion and quantitated using fluorescent labelling followed by HPLC analysis. PolySia expression was significantly increased following NGF, M3G or LPS stimulation when compared with unstimulated cells or cells exposed to the TLR4 antagonist LPS-RS. Additionally, we analyzed the effects of test agent exposure on cell migration and the oxidative stress response of these cells in the presence and absence of polySia expression on their cell surface. We observed an increase in oxidative stress in cells without polySia as well as following M3G or LPS stimulation. Our study provides evidence that polySia expression in neuronal-like PC12 cells is influenced by M3G and LPS exposure alike, suggestive of a role of TLR4 in triggering these events.

Time-resolved microfluidic flow cytometer for decoding luminescence lifetimes in the microsecond region.

Time-resolved luminescence detection using long-lived probes with lifetimes in the microsecond region have shown great potential in ultrasensitive and multiplexed bioanalysis. In flow cytometry, however, the long lifetime poses a significant challenge to measure wherein the detection window is often too short to determine the decay characteristics. Here we report a time-resolved microfluidic flow cytometer (tr-mFCM) incorporating an acoustic-focusing chip, which allows slowing down of the flow while providing the same detection conditions for every target, achieving accurate lifetime measurement free of autofluorescence interference. Through configuration of the flow velocity and detection aperture with respect to the time-gating sequence, a multi-cycle luminescence decay profile is captured for every event under maximum excitation and detection efficiency. A custom fitting algorithm is then developed to resolve europium-stained polymer microspheres as well as leukemia cells against abundant fluorescent particles, achieving counting efficiency approaching 100% and lifetime CVs (coefficient of variation) around 2-6%. We further demonstrate lifetime-multiplexed detection of prostate and bladder cancer cells stained with different europium probes. Our acoustic-focusing tr-mFCM offers a practical technique for rapid screening of biofluidic samples containing multiple cell types, especially in resource-limited environments such as regional and/or underdeveloped areas as well as for point-of-care applications.

Rapid and Label-Free Isolation of Tumour Cells from the Urine of Patients with Localised Prostate Cancer Using Inertial Microfluidics.

During the last decade, isolation of circulating tumour cells via blood liquid biopsy of prostate cancer (PCa) has attracted significant attention as an alternative, or substitute, to conventional diagnostic tests. However, it was previously determined that localised forms of PCa shed a small number of cancer cells into the bloodstream, and a large volume of blood is required just for a single test, which is impractical. To address this issue, urine has been used as an alternative to blood for liquid biopsy as a truly non-invasive, patient-friendly test. To this end, we developed a spiral microfluidic chip capable of isolating PCa cells from the urine of PCa patients. Potential clinical utility of the chip was demonstrated using anti-Glypican-1 (GPC-1) antibody as a model of the primary antibody in immunofluorescent assay for identification and detection of the collected tumour cells. The microchannel device was first evaluated using DU-145 cells in a diluted Dulbecco's phosphate-buffered saline sample, where it demonstrated >85 (±6) % efficiency. The microchannel proved to be functional in at least 79% of cases for capturing GPC1+ putative tumour cells from the urine of patients with localised PCa. More importantly, a correlation was found between the amount of the captured GPC1+ cells and crucial diagnostic and prognostic parameter of localised PCa-Gleason score. Thus, the technique demonstrated promise for further assessment of its diagnostic value in PCa detection, diagnosis, and prognosis.

Reduced background autofluorescence for cell imaging using nanodiamonds and lanthanide chelates.

Bio-imaging is a key technique in tracking and monitoring important biological processes and fundamental biomolecular interactions, however the interference of background autofluorescence with targeted fluorophores is problematic for many bio-imaging applications. This study reports on two novel methods for reducing interference with cellular autofluorescence for bio-imaging. The first method uses fluorescent nanodiamonds (FNDs), containing nitrogen vacancy centers. FNDs emit at near-infrared wavelengths typically higher than most cellular autofluorescence; and when appropriately functionalized, can be used for background-free imaging of targeted biomolecules. The second method uses europium-chelating tags with long fluorescence lifetimes. These europium-chelating tags enhance background-free imaging due to the short fluorescent lifetimes of cellular autofluorescence. In this study, we used both methods to target E-selectin, a transmembrane glycoprotein that is activated by inflammation, to demonstrate background-free fluorescent staining in fixed endothelial cells. Our findings indicate that both FND and Europium based staining can improve fluorescent bio-imaging capabilities by reducing competition with cellular autofluorescence. 30 nm nanodiamonds coated with the E-selectin antibody was found to enable the most sensitive detective of E-selectin in inflamed cells, with a 40-fold increase in intensity detected.

"Turn-on" Fluorescent Aptasensor Based on AIEgen Labeling for the Localization of IFN-γ in Live Cells.

We report an aggregation-induced emission fluorogen (AIEgen)-based turn-on fluorescent aptasensor able to detect the ultrasmall concentration of intracellular IFN-γ. The aptasensor consists of an IFN-γ aptamer labeled with a fluorogen with a typical aggregation-induced emission (AIE) characteristic, which shows strong red emission only in the presence of IFN-γ. The aptasensor is able to effectively monitor intracellular IFN-γ secretion with the lowest detection limit of 2 pg mL-1, and it is capable of localizing IFN-γ in live cells during secretion, with excellent cellular permeability and biocompatibility as well as low cytotoxicity. This probe is able to localize the intracellular IFN-γ at a low concentration <10 pg mL-1, and it is successfully used for real-time bioimaging. This simple and highly sensitive sensor may enable the exploration of cytokine pathways and their dynamic secretion process in the cellular environment. It provides a universal sensing platform for monitoring a spectrum of molecules secreted by cells.

A novel biocompatible europium ligand for sensitive time-gated immunodetection.

We describe the synthesis of a novel hydrophilic derivative of a tetradentate ß-diketone europium ligand that was used to prepare an immunoconjugate probe against Giardia lamblia cysts. We used a Gated Autosynchronous Luminescence Detector (GALD) to obtain high quality delayed luminescence images of cells 30-fold faster than ever previously reported.

A Novel Universal Detection Agent for Time-Gated Luminescence Bioimaging.

Luminescent lanthanide chelates have been used to label antibodies in time-gated luminescence (TGL) bioimaging. However, it is a challenging task to label directly an antibody with lanthanide-binding ligands and achieve control of the target ligand/protein ratios whilst ensuring that affinity and avidity of the antibody remain uncompromised. We report the development of a new indirect detection reagent to label antibodies with detectable luminescence that circumvents this problem by labelling available lysine residues in the linker portion of the recombinant fusion protein Linker-Protein G (LPG). Succinimide-activated lanthanide chelating ligands were attached to lysine residues in LPG and Protein G (without Linker) and the resulting Luminescence-Activating (LA-) conjugates were compared for total incorporation and conjugation efficiency. A higher and more efficient incorporation of ligands at three different molar ratios was observed for LPG and this effect was attributed to the presence of eight readily available lysine residues in the linker region of LPG. These Luminescence-Activating (LA-) complexes were subsequently shown to impart luminescence (upon formation of europium(III) complexes) to cell-specific antibodies within seconds and without the need for any complicated bioconjugation procedures. The potential of this technology was demonstrated by direct labelling of Giardia cysts and Cryptosporidium oocysts in TGL bioimaging.

Stable Upconversion Nanohybrid Particles for Specific Prostate Cancer Cell Immunodetection.

Prostate cancer is one of the male killing diseases and early detection of prostate cancer is the key for better treatment and lower cost. However, the number of prostate cancer cells is low at the early stage, so it is very challenging to detect. In this study, we successfully designed and developed upconversion immune-nanohybrids (UINBs) with sustainable stability in a physiological environment, stable optical properties and highly specific targeting capability for early-stage prostate cancer cell detection. The developed UINBs were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS) and luminescence spectroscopy. The targeting function of the biotinylated antibody nanohybrids were confirmed by immunofluorescence assay and western blot analysis. The UINB system is able to specifically detect prostate cancer cells with stable and background-free luminescent signals for highly sensitive prostate cancer cell detection. This work demonstrates a versatile strategy to develop UCNPs based sustainably stable UINBs for sensitive diseased cell detection.

N,O-isopropylidenated threonines as tools for peptide cyclization: application to the synthesis of mahafacyclin B.

[structure: see text] The influence of a single N,O-isopropylidenated threonine turn-inducer on the cyclization of a linear heptapeptide precursor to mahafacyclin B has been investigated. Incorporation of an N,O-isopropylidenated threonine more than doubles the head-to-tail cyclization yield. The N,O-isopropylidene grouping is then readily disassembled to give the antimalarial cyclic peptide in high yield.

Fluorescence detection of Fe(3+) ions in aqueous solution and living cells based on a high selectivity and sensitivity chemosensor.

Although ferric ion (Fe(3+)) performs critical roles in diverse biochemical processes in living systems, its physiological and pathophysiological functions have not been fully explored due to the lack of methods for quantification of Fe(3+) ions in biological system. In this work, a highly sensitive and selective fluorescence chemosensor, L, was developed for the detection of Fe(3+) ions in aqueous solution and in living cells. L was facile synthesized by one step reaction and well characterized by NMR, API-ES, FT-IR, and elementary analysis. The prepared chemosensor displayed excellent selectivity for Fe(3+) ions detection over a wide range of tested metal ions. In the present of Fe(3+) ions, the strong green fluorescence of L was substantially quenched. The 1:1 stoichiometry of the complexation was confirmed by a Job's plot. The association constant (Ka) of L with Fe(3+) was evaluated using the Benesi-Hildebrand method and was found to be 1.36×10(4) M(-1). The MTT assay determined that L exhibits low cytotoxicity toward living cells. Confocal imaging and flow cytometry studies showed that L is readily interiorized by MDA-MB-231 cells through an energy-dependent pathway and could be used to detect of Fe(3+) ions in living cells.

Synthesis of all-L cyclic tetrapeptides using pseudoprolines as removable turn inducers.

Cyclic tetrapeptides have generated great interest because of their broad-ranging biological properties. In order to synthesize these highly strained 12-membered cyclic compounds, a cyclization strategy using pseudoprolines as removable turn inducers has been developed. The pseudoproline derivatives induce a cisoid amide bond in the linear peptide backbone which facilitates cyclization. After cyclization, the turn inducers can be readily removed to afford cyclic tetrapeptides containing serine or threonine residues.