Intracellular Detection Research Articles

Intracellular Amyloid-β Detection from Human Brain Sections Using a Microfluidic Immunoassay in Tandem with MALDI-MS.

Alzheimer's disease (AD) currently affects more than 30 million people worldwide. The lack of understanding of AD's physiopathology limits the development of therapeutic and diagnostic tools. Soluble amyloid-β peptide (Aβ) oligomers that appear as intermediates along the Aβ aggregation into plaques are considered among the main AD neurotoxic species. Although a wealth of data are available about Aβ from in vitro and animal models, there is little known about intracellular Aβ in human brain cells, mainly due to the lack of technology to assess the intracellular protein content. The elucidation of the Aβ species in specific brain cell subpopulations can provide insight into the role of Aβ in AD and the neurotoxic mechanism involved. Here, we report a microfluidic immunoassay for in situ mass spectrometry analysis of intracellular Aβ species from archived human brain tissue. This approach comprises the selective laser dissection of individual pyramidal cell bodies from tissues, their transfer to the microfluidic platform for sample processing on-chip, and mass spectrometric characterization. As a proof-of-principle, we demonstrate the detection of intracellular Aβ species from as few as 20 human brain cells.

Methylglyoxal-Modified Albumin Effects on Endothelial Arginase Enzyme and Vascular Function.

Advanced glycation end products (AGEs) contribute significantly to vascular dysfunction (VD) in diabetes. Decreased nitric oxide (NO) is a hallmark in VD. In endothelial cells, NO is produced by endothelial NO synthase (eNOS) from L-arginine. Arginase competes with NOS for L-arginine to produce urea and ornithine, limiting NO production. Arginase upregulation was reported in hyperglycemia; however, AGEs' role in arginase regulation is unknown. Here, we investigated the effects of methylglyoxal-modified albumin (MGA) on arginase activity and protein expression in mouse aortic endothelial cells (MAEC) and on vascular function in mice aortas. Exposure of MAEC to MGA increased arginase activity, which was abrogated by MEK/ERK1/2 inhibitor, p38 MAPK inhibitor, and ABH (arginase inhibitor). Immunodetection of arginase revealed MGA-induced protein expression for arginase I. In aortic rings, MGA pretreatment impaired acetylcholine (ACh)-induced vasorelaxation, which was reversed by ABH. Intracellular NO detection by DAF-2DA revealed blunted ACh-induced NO production with MGA treatment that was reversed by ABH. In conclusion, AGEs increase arginase activity probably through the ERK1/2/p38 MAPK pathway due to increased arginase I expression. Furthermore, AGEs impair vascular function that can be reversed by arginase inhibition. Therefore, AGEs may be pivotal in arginase deleterious effects in diabetic VD, providing a novel therapeutic target.

Protein-Scaffolded DNA Nanostructures for Imaging of Apurinic/Apyrimidinic Endonuclease 1 Activity in Live Cells.

Nucleic acids are valuable tools for intracellular biomarker detection and gene regulation. Here we propose a new type of protein (avidin)-scaffolded DNA nanostructure (ADN) for imaging the activity of apurinic/apyrimidinic endonuclease 1 (APE1) in live cells. ADN is designed by assembling an avidin-displayed abasic site containing DNA strands labeled with a fluorophore or a quencher via a complementary linker strand. ADN is nonemissive due to the close proximity of fluorophores and quenchers. APE1-mediated cleavage separates the fluorophores from the quenchers, delivering activated fluorescence. In vitro assays show that ADN is responsive to APE1 with high sensitivity and high specificity. ADN can efficiently enter the cells, and its capability to visualize and detect intracellular APE1 activities is demonstrated in drug-treated cells and different cell lines. The modular and easy preparation of our nanostructures would afford a valuable platform for imaging and detecting APE1 activities in live cells.

Structural and functional properties of the Kunitz-type and C-terminal domains of Amblyomin-X supporting its antitumor activity.

Amblyomin-X is a Kunitz-type FXa inhibitor identified through the transcriptome analysis of the salivary gland from Amblyomma sculptum tick. This protein consists of two domains of equivalent size, triggers apoptosis in different tumor cell lines, and promotes regression of tumor growth, and reduction of metastasis. To study the structural properties and functional roles of the N-terminal (N-ter) and C-terminal (C-ter) domains of Amblyomin-X, we synthesized them by solid-phase peptide synthesis, solved the X-Ray crystallographic structure of the N-ter domain, confirming its Kunitz-type signature, and studied their biological properties. We show here that the C-ter domain is responsible for the uptake of Amblyomin-X by tumor cells and highlight the ability of this domain to deliver intracellular cargo by the strong enhancement of the intracellular detection of molecules with low cellular-uptake efficiency (p15) after their coupling with the C-ter domain. In contrast, the N-ter Kunitz domain of Amblyomin-X is not capable of crossing through the cell membrane but is associated with tumor cell cytotoxicity when it is microinjected into the cells or fused to TAT cell-penetrating peptide. Additionally, we identify the minimum length C-terminal domain named F2C able to enter in the SK-MEL-28 cells and induces dynein chains gene expression modulation, a molecular motor that plays a role in the uptake and intracellular trafficking of Amblyomin-X.

Near-IR intracellular ratiometric ‘turn-on’ discrimination of H2S/Cys and low-cost test kits for ppm level detection of H2S gas

• Synthesis of bay -functionalized unsymmetric trisubstituted PDI 1 and 2 is reported. • Ratiometric ‘turn-on’ absorbance (A 688 /A 560 ); fluorescence (F 670 /F 526 ) changes with H 2 S. • Low-cost solution-, solid-based test kits for the detection of ppm level of H 2 S gas. • We successfully applied PDI 1 for the intracellular detection of H 2 S in live A549 cells. We report the sustainable design for intracellular ratiometric ‘ turn-on ’ detection and discrimination of gaseous H 2 S and Cys using bay-functionalized trisubstituted PDI 1 and 2 . The two nitro groups were incorporated on PDI 1 and 2 as an effective target site for H 2 S, whereas presence of O-propargyl (PDI 1 ) and O-butyn (PDI 2 ) may be effective sites for H 2 S at high concentrations. This detection approach showed ratiometric ‘ turn-on ’ absorbance (A 688 /A 560 ) and fluorescence (F 670 /F 526 ) response associated with visual colour change from purple to green and green/red emission in the NIR region upon addition of H 2 S. These results were supported by current-voltage (I–V) plot, AFM, DLS studies and further augmented by isolation and characterization of intermediate product of reaction between PDI 1 and H 2 S. The application of PDI 1 and 2 as solution-based test kits for detection of 37.5 ppm H 2 S gas with naked eye colour change from purple to green to blue in 20s; PDI 1 adsorbed silica and TLC strips as solid-state test kits for detection of 150 ppm and 300–600 ppm H 2 S gas, respectively in 5 mins and exogenous detection of 150 and 750 µM H 2 S in A549 live cells have been demonstrated.

Real-time detection of bone-invasive oral cancer with laser-induced breakdown spectroscopy: A proof-of-principle study

ObjectivesIntraoperative definition of resection margin status in bone-invasive oral cancer is a fundamental problem in oncologic surgery due to the lack of rapid bone analysis methods. Laser-induced breakdown spectroscopy (LIBS) provides direct measurement with real-time examination of a minimal tissue sample. This proof-of-principle study aimed to evaluate the possibility of distinguishing tumorous and healthy areas with LIBS. Materials and MethodsLIBS experiments were executed on native segmental mandibulectomy specimens from 15 patients with bone-invasive oral cancer. Normalized and intensity-matched spectra were compared. Under biological derivation, peak area calculation and principal component analysis (PCA) were applied. The discriminatory power of the PCAs was correlated with the architectural and cytological characteristics of the lasered tumor tissue. Receiver operating characteristics analysis was used to evaluate the performance of LIBS in the real-time detection of bone-invasive cancer. ResultsCalcium (Ca), which is high in healthy bone, is replaced by potassium (K) and sodium (Na) in bone-invasive cancer. The degree of stromal induction is significantly correlated with the discriminatory power between healthy and tumorous spectra. In this study, LIBS ensured an overall sensitivity of 95.51% and a specificity of 98.64% via the intracellular detection of K and Na. ConclusionThis study demonstrated robust real-time detection of bone-invasive oral cancer with LIBS, which may lay the foundation for establishing LIBS as a rapid bone analysis method. Further development of a LIBS-guided assessment of bone tumor resection margins might reduce the extent of bony resection without compromising oncologic safety.

Catalyst-Accelerated Circular Cascaded DNA Circuits: Simpler Design, Faster Speed, Higher Gain.

DNA cascaded circuits have great potential in detecting low abundance molecules in complex biological environment due to their powerful signal amplification capability and nonenzymatic feature. However, the problem of the cascaded circuits is that the design is relatively complex and the kinetics is slow. Herein, a new design paradigm called catalyst-accelerated circular cascaded circuits is proposed, where the catalyst inlet is implanted and the reaction speed can be adjusted by the catalyst concentration. This new design is very simple and only requires three hairpin probes. Meanwhile, the results of a series of studies demonstrate that the reaction speed can be accelerated and the sensitivity can be also improved. Moreover, endogenous mRNA can also be used as a catalyst to drive the circuits to amplify the detection of target miRNA in live cells and in mice. These catalyst-accelerated circular cascaded circuits can substantially expand the toolbox for intracellular low abundance molecular detection.

A 1,8-naphthalimide based chemosensor for intracellular and biofluid detection of Pd2+ ions: microscopic and anticounterfeiting studies.

A naphthalimide based chemosensor (NPG), containing 1,8-naphthalimide as a fluorophore unit and pentaethylene glycol as a binding unit, has been used for the detection of Pd2+ ions in 50% HEPES buffer-DMSO (pH 7.2) solution. The NPG showed aggregation induced emission enhancement (AIEE) properties in H2O-DMSO binary mixtures (0-90%) and the CIE plot of NPG in DMSO has x = 0.152, y = 0.102 coordinates corresponding to blue colour emission with 86% colour purity. Upon addition of Pd2+ ions, NPG showed a decrease in fluorescence intensity associated with a colour change from fluorescent blue to non-fluorescent colourless solution. The lowest limit of detection for Pd2+ ions was 75 nM. The mechanism of interaction of NPG with Pd2+ ions leads to complexation induced aggregation caused quenching (ACQ) supported by DLS, SEM and AFM studies. The NPG has been successfully utilized for (i) intracellular detection of Pd2+ ions (250 μM) in live MG-63 cells; (ii) detection of Pd2+ ions in pharmaceutical (99.74 ± 0.6%), urine (98.20 ± 2.96%) and blood serum (99.17 ± 1.84%) samples and (iii) detection of Pd2+ ions using silica coated TLC strips via a contact mode method. NPG can be used as a security ink for writing letters and alphabets for anticounterfeiting applications.

Metformin enhances the osteogenic activity of rat bone marrow mesenchymal stem cells by inhibiting oxidative stress induced by diabetes mellitus: an in vitro and in vivo study.

The purpose of this study was to determine whether metformin (MF) could alleviate the expresssion of reactive oxygen species (ROS) and improve the osteogenic ability of bone marrow mesenchymal stem cells derived from diabetic rats (drBMSCs) in vitro, and to evaluate the effect of MF on the ectopic osteogenesis of drBMSCs in a nude mouse model in vivo. BMSCs were extracted from normal and diabetic rats. In vitro, a cell viability assay (Cell Counting Kit-8), tests of alkaline phosphatase (ALP) activity, and western blot analysis were first used to determine the cell proliferation and osteogenic differentiation of drBMSCs that were subjected to treatment with different concentrations of MF (0, 50, 100, 200, 500 μM). The cells were then divided into 5 groups: (1) normal rat BMSCs (the BMSCs derived from normal rats group), (2) the drBMSCs group, (3) the drBMSCs + Mito-TEMPO (10 μM, ROS scavenger) group, (4) the drBMSCs + MF (200 μM) group, and (5) the drBMSCs + MF (200 μM) + H2O2 (50 μM, ROS activator) group. Intracellular ROS detection, a senescence-associated β-galactosidase assay, ALP staining, alizarin red staining, western blotting, and immunofluorescence assays were performed to determine the effects of MF on oxidative stress and osteogenic differentiation in drBMSCs. In vivo, the effect of MF on the ectopic osteogenesis of drBMSCs was evaluated in a nude mouse model. MF effectively reduced ROS levels in drBMSCs. The cell proliferation, ALP activity, mineral deposition, and osteogenic-related protein expression of drBMSCs were demonstrably higher in the MF-treated group than in the non-MF-treated group. H2O2 inhibited the effects of MF. In addition, ectopic osteogenesis was significantly increased in drBMSCs treated with MF. MF promoted the proliferation and osteogenic differentiation of drBMSCs by inhibiting the oxidative stress induced by diabetes and enhenced the ectopic bone formation of drBMSCs in nude mice.

Phosphoric acid densified red emissive carbon dots with a well-defined structure and narrow band fluorescence for intracellular reactive oxygen species detection and scavenging

Phosphoric acid-densified red emissive carbon dots (R-CDs) with well-defined structures and narrow band fluorescence were prepared for the detection of ROS under pathological conditions.

Fluorogenic Probes for Intracellular Iron Detection.

Iron is a crucial element required to sustain multiple biological processes, including oxygen transport, DNA synthesis, and electron transport. In living cells, iron exists as either ferrous iron (Fe2+) or ferric iron (Fe3+), and its redox forms are regulated by the labile iron pool. Both iron deficiency and excess can lead to a range of pathological conditions, such as anemia, cancer, neurodegenerative disorders, and ischemia and reperfusion injury. Iron overload can cause oxidative damage and even cell death, especially via ferroptosis. Impaired ferroptosis pathways are implicated in the pathogenesis of various diseases and are becoming attractive therapeutic targets. Therefore, developing methods to analyze dynamic iron changes in cells is crucial. In this chapter, we introduce several protocols that use fluorogenic iron probes (e.g., FerroFarRed, Calcein-AM, and FRET iron probe 1) to measure intracellular iron content.

Chitosan functionalized gold nanostars as a theranostic platform for intracellular microRNA detection and photothermal therapy.

The development of a theranostic platform that integrates both diagnostic and therapeutic capabilities is in great need for precise and personalized medicine. Here, we present a novel nanoplatform (AuNS@CS-hpDNA) formulated by chitosan functionalized gold nanostar composites and further complexed with fluorescent hairpin DNA (hpDNA) probes for tumor-related miRNA imaging and photothermal therapy (PTT). The optimized AuNS@CS-hpDNA nanoplatform mediated efficient hpDNA probe loading and intracellular delivery. Subsequently, the cytosol transfer of the hpDNA probe enabled specific hybridization using the targeted miRNA, which triggered the recovery of fluorescence for the precise detection of biomarker miR21 in living cells and realized the distinguishing cancer cell line MCF-7 and normal cells. Meanwhile, the AuNS@CS-hpDNA nanoplatform exhibited excellent photothermal conversion properties, which induced efficient cancer cell killing under laser irradiation. Thus, the developed AuNS@CS-hpDNA nanoplatform could simultaneously realize the precise detection of cancer cells and accurately initiate efficient PTT, which represents a promising strategy for precise cancer therapy.

Assessing metal ion transporting activity of ZIPs: Intracellular zinc and iron detection.

Zrt/Irt-like proteins (ZIPs or SLC39A) are a large family of metal ion transporters mainly responsible for zinc uptake. Some ZIPs have been shown to specifically transport zinc, whereas others have broader substrate specificity in divalent metal ion trafficking, notably those of zinc and iron ions. Measuring intracellular zinc and iron levels helps assess their molecular and physiological activities. This chapter presents step-by-step methods for evaluating intracellular metal ion concentrations, including direct measurement using inductively coupled plasma-mass spectrometry (ICP-MS), chemical staining, fluorescent probes, and indirect reporter assays such as activity analysis of enzymes whose activities are dependent on metal ion availability.

Binding affinity-based intracellular drug detection enabled by a unimolecular cucurbit[7]uril-dye conjugate.

Label-free fluorescence-based chemosensing has been increasingly brought into focus due to its simplicity and high sensitivity for intracellular monitoring of molecules. Currently used methods, such as conventional indicator displacement assays (IDAs), pose limitations related to dissociation upon dilution, random diffusion of the released indicators, and high sensitivity to interference by agents from the ambient cellular environment (e.g., salts, enzymes, and proteins). Herein we report a potentially widely applicable strategy to overcome the limitations of conventional IDAs by employing a macrocyclic cucurbit[7]uril (CB7) host covalently coupled to a nitrobenzoxadiazole (NBD) fluorescent dye (CB7-NBD conjugate). As a proof of concept, we demonstrated that the CB7-NBD unimolecular conjugate responded to various target analytes even in the complex live cell system. Moreover, the sensing system was compatible with fluorescence imaging, fluorescence-assisted cell sorting (FACS), and fluorescence spectrometry with a microplate reader. These experiments demonstrated an application of covalently bound unimolecular CB7-NBD conjugate as a sensor for detecting diverse analytes in the intracellular compartment of live cells.

Abstract 1955: Intracellular SERS detection of nanoparticle drug delivery platforms

Abstract 1955: Intracellular SERS detection of nanoparticle drug delivery platforms

On the validity of fluorimetric intracellular calcium detection: Impact of lipid components

We investigated the effects of different lipids on the activity of the angiotensin II type 1 receptor (AT1R). As calcium plays a key role in the signaling of the AT1R, we used the calcium-sensitive fluorescence indicators fura-2 to detect intracellular calcium release upon stimulation with the agonist angiotensin II. At first sight, cells preincubated with Very low-density lipoprotein (VLDL) showed a reduced calcium release triggered by angiontensin II compared to untreated control. However, on closer examination, this result seemed to be an artifact. Incubation with VLDL reduced also the amount of intracellular fura-2, as measured by fluorescence in the isosbestic point. Additionally, the maximal obtainable ratio, obtained after complete saturation with calcium ions, was reduced in cells preincubated with VLDL. These findings rendered our initial results questionable. We report the results of our work and our suggestions regarding the experimental setup to contribute to the understanding of the interpretation of fura-2 measurements and to avoid erroneous conclusions.

Dual-emissive carbonized polymer dots for the ratiometric fluorescence imaging of singlet oxygen in living cells

Singlet oxygen (1O2) is a type of reactive oxygen species (ROS), playing a vital role in the physiological and pathophysiological processes. Specific probes for monitoring intracellular 1O2 still remain challenging. In this study, we develop a ratiometric fluorescent probe for the real-time intracellular detection of 1O2 using o-phenylenediamine-derived carbonized polymer dots (o-PD CPDs). The o-PD CPDs possessing dual-excitation-emission properties (blue and yellow fluorescence) were successfully synthesized in a two-phase system (water/acetonitrile) using an ionic liquid tetrabutylammonium hexafluorophosphate as a supporting electrolyte through the electrolysis of o-PD. The o-PD CPDs can act as a photosensitizer to produce 1O2 upon white LED irradiation, in turn, the generated 1O2 selectively quenches the yellow emission of the o-PD CPDs. This quenching behavior is ascribed to the specific cycloaddition reaction between 1O2 and alkene groups in the polymer scaffolds on o-PD CPDs. The interior carbon core can be a reliable internal standard since its blue fluorescence intensity remains unchanged in the presence of 1O2. The ratiometric response of o-PD CPDs is selective toward 1O2 against other ROS species. The developed o-PD CPDs have been successfully applied to monitor the 1O2 level in the intracellular environment. Furthermore, in the inflammatory neutrophil cell model, o-PD CPDs can also detect the 1O2 and other ROS species such as hypochlorous acid after phorbol 12-myristate 13-acetate (PMA)-induced inflammation. Through the dual-channel fluorescence imaging, the ratiometric response of o-PD CPDs shows great potential for detecting endogenous and stimulating 1O2in vivo.

Novel Green Fluorescent Probe Stem From Carbon Quantum Dots for Specific Recognition of Tyrosinase in Serum and Living Cells.

Tyrosinase (TYR), an important biomarker for melanoma, offered significant information early detection of melanoma and may decrease the likelihood of mortality. Therefore, this article constructed a highly sensitive and selective green fluorescent functionalized carbon quantum dots (TYR-CQDs) for tyrosinase (TYR) activity detection by one-step hydrothermal protocol utilizing catechol, citric acid and urea as precursors. The prepared TYR-CQDs illustrated excellent linear relationship and broad linear range with a low detection limit, which exhibited high accuracy and recovery in quantitative determination of TYR in human serum samples. Furthermore, the TYR-CQDs had successfully realized intracellular TYR detection owing to excellent biocompatibility, high anti-interference ability and good cellular imaging capability, suggesting the potential biomedical applications in early diagnosis of melanoma and other tyrosinase-related diseases.

A Blue/NIR ratiometric fluorescent probe for intracellular detection of Tyrosinase and the inhibitor screening

A novel ratiometric fluorescent tyrosinase assay is developed based on hybrid nano-assembly of gold nanocluster and tyrosine-containing peptides. The AuNCs@YCY nano-probe (AYNP) is fabricated through the hydrophobic interactions and π-π stacking between the tyrosine residues of the Tyr-Cys-Tyr tripeptide (YCY) and the ligands on the surfaces of AuNCs under the near-isoelectric pH value. The resulted AYNP shows distinct fluorescence responses, spontaneous turn-on of the blue emission and turn-off of the near-infrared emission, with a single wavelength excitation. It is demonstrated that the enhancement and quenching are due to the production of pheomelanin and dopaquinone structures, respectively, induced by tyrosinase oxidation. The internal referencing system provides the tyrosinase assay with superior sensitivity and a detection limit as low as 6.3 U L−1 could be achieved. The experimental results also demonstrate the excellent selectivity, good photo-stability, and both in vitro and cellular applications of AYNP. This assay technique is low-cost, easy to prepare, and shows excellent potential as a novel melanoma clinical diagnostic platform and a tyrosinase inhibitor screening tool.

DNAzymes-conjugated upconversion nanoamplicon for in-situ ultrasensitive detection and imaging of microRNA in vivo

Upconversion nanoparticles (UCNPs) are promising materials for imaging biomolecules and predicting the progression of diseases, especially for tumors. However, conventional luminescence resonance energy transfer (LRET) based nanosensors remain challenging with complicated modification and unsatisfactory sensitivity. Herein, a sensing platform integrating confined emission effect and cycling amplification strategy into one substrate with high LRET efficiency and sensitivity has been developed for the in-vitro and in-vivo detection of microRNA-21. Washing UCNPs with hydrochloric acid was conducted for electrostatic absorption of 8–17 DNAzyme labeled with BHQ1 (8–17 DNAzyme-BHQ1) and complementary capturing template-aDNA3 complex (CPT-aDNA3) to form energy donor–acceptor pair (UCNPs-BHQ1) and capture miR-21. It not only avoids the complex modification procedure but also shortens the distance between the donor (UCNPs) and the acceptor (BHQ1). The quenching efficiency of 60.94 % was obtained. Targeted miR-21 could bind with CPT to release aDNA3 that can unlock 8–17 DNAzyme-BHQ1 to form aDNA3-DNAzyme-BHQ1 duplex. This aDNA3-DNAzyme-BHQ1 duplex owning -rA- group can be cleaved by Zn2+ so that UCNPs restored green UCL emission because of the desorption of BHQ1. aDNA3 becomes dissociative again to trigger the next cycle. This UCNP@DNAzyme-BHQ1/CPT- aDNA3 achieved a wide linear range from 4 × 10-14 M to 1 × 10-8 M with a low limit detection of 31 fM for the sensitive detection of miR-21 in vitro. Furthermore, it was successfully applied to the real-time intracellular detection of miR-21 in HeLa cells and in-vivo monitoring of miR-21 expression in KS-01 treated tumor-bearing mouse. This proposed DNA-conjugated upconversion nanoamplicon provides new sights into monitoring of anti-cancer active ingredients efficacy and the progress evaluation of miRNA-related diseases.