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1.
Front Neurosci ; 18: 1408087, 2024.
Article in English | MEDLINE | ID: mdl-38962178

ABSTRACT

Vision plays a major role in perceiving external stimuli and information in our daily lives. The neural mechanism of color vision is complicated, involving the co-ordinated functions of a variety of cells, such as retinal cells and lateral geniculate nucleus cells, as well as multiple levels of the visual cortex. In this work, we reviewed the history of experimental and theoretical studies on this issue, from the fundamental functions of the individual cells of the visual system to the coding in the transmission of neural signals and sophisticated brain processes at different levels. We discuss various hypotheses, models, and theories related to the color vision mechanism and present some suggestions for developing novel implanted devices that may help restore color vision in visually impaired people or introduce artificial color vision to those who need it.

2.
ACS Appl Mater Interfaces ; 16(20): 25879-25891, 2024 May 22.
Article in English | MEDLINE | ID: mdl-38718301

ABSTRACT

Biological imaging-guided targeted tumor therapy has been a soughtafter goal in the field of cancer diagnosis and treatment. To this end, we proposed a strategy to modulate surface plasmon resonance and endow WO3-x nanoparticles (NPs) with enzyme-like catalytic properties by doping Fe2+ in the structure of the NPs. Doping of the Fe2+ introduced oxygen vacancies into the structure of the NPs, inducing a red shift of the maximum absorption wavelength into the near-infrared II (NIR-II) region and enhancing the photoacoustic (PA) and photothermal properties of the NPs for more effective imaging-guided cancer therapy. Under NIR-II laser irradiation, the Fe-WO3-x NPs produced very strong NIR-II PA and photothermal effects, which significantly enhanced the PA imaging and photothermal treatment effects. On the other hand, Fe2+ in Fe-WO3-x could undergo Fenton reactions with H2O2 in the tumor tissue to generate ·OH for chemodynamic therapy. In addition, Fe-WO3-x can also catalyze the above reactions to produce more reactive oxygen species (ROS) and induce the oxidation of NADH to interfere with intracellular adenosine triphosphate (ATP) synthesis, thereby further improving the efficiency of cancer therapy. Specific imaging of tumor tissue and targeted synergistic therapy was achieved after ligation of a MUC1 aptamer to the surface of the Fe-WO3-x NPs by the complexing of -COOH in MUC1 with tungsten ions on the surface of the NPs. These results demonstrated that Fe-WO3-x NPs could be a promising diagnosis and therapeutic agent for cancer. Such a study opens up new avenues into the rational design of nanodiagnosis and treatment agents for NIR-II PA imaging and cancer therapy.


Subject(s)
Photoacoustic Techniques , Surface Plasmon Resonance , Tungsten , Animals , Humans , Mice , Tungsten/chemistry , Infrared Rays , Oxides/chemistry , Neoplasms/diagnostic imaging , Neoplasms/therapy , Neoplasms/drug therapy , Nanoparticles/chemistry , Nanoparticles/therapeutic use , Cell Line, Tumor , Reactive Oxygen Species/metabolism
3.
Anal Chem ; 96(1): 85-91, 2024 01 09.
Article in English | MEDLINE | ID: mdl-38128902

ABSTRACT

Cellular trace proteins are critical for maintaining normal cell functions, with their quantitative analysis in individual cells aiding our understanding of the role of cell proteins in biological processes. This study proposes a strategy for the quantitative analysis of alpha-fetoprotein in single cells, utilizing a lysosome microenvironment initiation and a DNAzyme-assisted intracellular signal amplification technique based on electrophoretic separation. A nanoprobe targeting lysosomes was prepared, facilitating the intracellular signal amplification of alpha-fetoprotein. Following intracellular signal amplification, the levels of alpha-fetoprotein (AFP) in 20 HepG2 hepatoma cells and 20 normal HL-7702 hepatocytes were individually evaluated using microchip electrophoresis with laser-induced fluorescence detection (MCE-LIF). Results demonstrated overexpression of alpha-fetoprotein in hepatocellular carcinoma cells. This strategy represents a novel technique for single-cell protein analysis and holds significant potential as a powerful tool for such analyses.


Subject(s)
Carcinoma, Hepatocellular , DNA, Catalytic , Electrophoresis, Microchip , Liver Neoplasms , Humans , alpha-Fetoproteins/analysis , Electrophoresis, Microchip/methods , Nucleic Acid Amplification Techniques/methods , Lysosomes/chemistry , Carcinoma, Hepatocellular/pathology , Tumor Microenvironment
4.
Adv Sci (Weinh) ; 11(11): e2306375, 2024 Mar.
Article in English | MEDLINE | ID: mdl-38161215

ABSTRACT

Tumor metastasis remains a leading factor in the failure of cancer treatments and patient mortality. To address this, a silver-induced absorption red-shifted core-shell nano-particle is developed, and surface-modified with triphenylphosphonium bromide (TPP) and hyaluronic acid (HA) to obtain a novel nanodiagnosis-treatment agent (Ag@CuS-TPP@HA). This diagnosis-treatment agent can dual-targets cancer cells and mitochondria, and exhibits maximal light absorption at 1064 nm, thereby enhancing nesr-infrared II (NIR-II) photoacoustic (PA) signal and photothermal effects under 1064 nm laser irradiation. Additionally, the silver in Ag@CuS-TPP@HA can catalyze the Fenton-like reactions with H2 O2 in the tumor tissue, yielding reactive oxygen species (ROS). The ROS production, coupled with enhanced photothermal effects, instigates immunogenic cell death (ICD), leading to a substantial release of tumor-associated antigens (TAAs) and damage-associated molecular patterns, which have improved the tumor immune suppression microenvironment and boosting immune checkpoint blockade therapy, thus stimulating a systemic antitumor immune response. Hence, Ag@CuS-TPP@HA, as a cancer diagnostic-treatment agent, not only accomplishes targeted the NIR-II PA imaging of tumor tissue and addresses the challenge of accurate diagnosis of deep cancer tissue in vivo, but it also leverages ROS/photothermal therapy to enhance immune checkpoint blockade, thereby eliminating primary tumors and effectively inhibiting distant tumor growth.


Subject(s)
Antineoplastic Agents , Neoplasms , Organophosphorus Compounds , Photoacoustic Techniques , Humans , Reactive Oxygen Species/metabolism , Silver , Immune Checkpoint Inhibitors , Photoacoustic Techniques/methods , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Neoplasms/therapy , Tumor Microenvironment
5.
Anal Chem ; 95(29): 11061-11069, 2023 07 25.
Article in English | MEDLINE | ID: mdl-37439625

ABSTRACT

The simultaneous quantification of multiple microRNAs (miRNA) in a single cell can help scientists understand the relationship between different miRNA groups and different types of cancers from an miRNA omics perspective at the single-cell level. However, there currently remains a challenge in developing techniques for the simultaneous absolute quantification of multiple miRNAs in single cells. Herein, we propose a framework nucleic acid (FNA)-mediated multimodal tandem multivariate signal amplification strategy for simultaneous absolute quantification of three different miRNAs in a single cell. In this study, DNA hexahedron FNAs (DHFs) and DNA tetrahedron FNAs (DTFs) were first prepared, multiple DNA hairpins and substrates were then connected to the hexahedron frame nucleic acid as the target recognition units, and three substrates with labeled FAM fluorophores on the tetrahedral frame nucleic acid served as signal output units. After the two types of FNAs entered the cell, they reacted with three different miRNAs (miRNA-155, miRNA-373, and miRNA-21) and multimodal tandem multivariate signal amplification was initiated simultaneously, reducing the detection limit of the three miRNAs to 8 × 10-15, 2 × 10-15, and 1 × 10-15 M, respectively. The detection sensitivity of the three miRNAs was simultaneously increased by six orders of magnitude, reaching the quantitative requirement of trace miRNAs in single cells. Combined with single-cell injection, membrane melting, and intracellular component separation technology on a microchip electrophoresis platform, we achieved the simultaneous absolute quantification of three different miRNAs in a single cell, thereby providing an important novel method that can be used to conduct single-cell research.


Subject(s)
MicroRNAs , Nucleic Acids , MicroRNAs/analysis , DNA/genetics , Fluorescent Dyes , Nucleic Acid Amplification Techniques/methods
6.
Anal Chem ; 94(50): 17645-17652, 2022 12 20.
Article in English | MEDLINE | ID: mdl-36475450

ABSTRACT

Slow intermolecular collisions and "always active" responses compromise the amplification efficiency and response accuracy of nonenzymatic hybridization chain reaction (HCR). In this study, a photoactivatable membrane-oriented HCR (MOHCR) system was rationally designed by binding a photocleavable initiator probe onto a target protein and then anchoring cholesterol-modified hairpin-structure fuel probes. When irradiated, the bound initiator probe was photoactivated and initiated self-assembly to generate activatable and amplified imaging. In a proof-of-concept assay, breast-cancer-derived exosomes were imaged based on the surface protein epithelial cell adhesion molecule (EpCAM). Photoactivatable responses provided precise spatiotemporal control of the MOHCR, and fluidic membranes enabled accelerated reaction kinetics. Our MOHCR system demonstrated high efficiency and accuracy in differentiating between plasma samples from breast cancer patients and healthy donors.


Subject(s)
Biosensing Techniques , Exosomes , Neoplasms , Humans , Exosomes/chemistry , Kinetics , Nucleic Acid Hybridization , Proteins/analysis , Biosensing Techniques/methods
7.
Anal Chem ; 94(45): 15847-15855, 2022 11 15.
Article in English | MEDLINE | ID: mdl-36327212

ABSTRACT

Single-cell protein analysis is very important for understanding cellular heterogeneity and single-cell biology. However, owing to the extremely low levels of some tumor-associated proteins in individual cells, the absolute quantification of such tumor-associated proteins in a single cell remains a challenge. Herein, an intracellular multicomponent synchronous DNA-walking strategy is proposed for the simultaneous quantification of multiple tumor-associated proteins in a single cell. In this strategy, a nanoprobe based on a DNA walker was designed for the simultaneous signal amplification of nucleolin (NCL) and thymidine kinase 1 (TK1) in a single cell. NCL and TK1 in single cells were simultaneously detected on a microchip platform with detection limits of 1.0 and 0.8 pM, respectively. The results obtained from 20 liver cancer cells (HepG2) and 20 normal hepatocytes (HL-7702) indicated that NCL and TK1 were overexpressed in liver cancer cells. However, the levels of NCL and TK1 in normal hepatocytes are only about one-tenth to one-sixth of those in hepatic carcinoma. Using different nucleic acid aptamers, the proposed strategy can be applied for the analysis of other single-cell proteins and in the early diagnosis of cancer.


Subject(s)
DNA , Liver Neoplasms , Humans , DNA/genetics , Neoplasm Proteins , Liver Neoplasms/diagnosis , Walking
8.
Biosens Bioelectron ; 217: 114702, 2022 Dec 01.
Article in English | MEDLINE | ID: mdl-36130443

ABSTRACT

Messenger ribonucleic acids (mRNAs) comprise a class of small nucleic acids carrying genetic information, which exhibit very important role in medical research and diagnosis. If only the mean mRNA expression levels of the mRNA population are considered in medical research, important information linking mRNA expression and cellular function may be lost. Single-cell analysis provides valuable insights into studying its heterogeneity, signaling, and stochastic gene expression. In this study, a "bunge bedstraw herb"-type DNA machine based on DNAzyme catalyzing coupled clamping hybrid chain reaction (c-HCR) is presented. In the DNA machine, a bunge bedstraw herb-type DNA structure was first formed by hybridizing a core junction scaffold cruciform probe to a hairpin probe that can trigger the c-HCR via a target molecule in four directions. This approach can reduce the detection limit of mRNA to 5 × 10-15 M. Absolute quantification of survivin mRNA in individual cells was achieved using the DNA machine on a microfluidic chip electrophoresis platform. The reported method represents an unprecedented single-cell analysis platform for single-cell biology studies.


Subject(s)
Biosensing Techniques , DNA, Catalytic , Galium , Biosensing Techniques/methods , DNA/chemistry , DNA/genetics , DNA, Catalytic/chemistry , Galium/genetics , Limit of Detection , Nucleic Acid Amplification Techniques/methods , Nucleic Acid Hybridization/methods , RNA, Messenger/analysis , RNA, Messenger/genetics , Survivin/genetics
9.
Biosens Bioelectron ; 203: 114053, 2022 May 01.
Article in English | MEDLINE | ID: mdl-35121443

ABSTRACT

MicroRNAs (miRNAs) play a very important regulatory role in life activities. Abnormal expression levels of miRNAs in cells are associated with various diseases, especially human cancer. Nevertheless, accurate detection of the copy numbers of various miRNA molecules in single cell is still a great challenge. In this study, an intracellular multivariate signal amplification strategy based on microchip platform was proposed, and an ultrasensitive single-cell analysis method was established for simultaneous quantification of absolute copy numbers of multiple miRNAs in a single cell. Using miRNA-21 and miRNA-141 as the analytical models of miRNAs, the detection limits of 1.0 and 2.0 fM were obtained. Based on the developed method, an analysis of 600 randomly acquired different types of cells was performed. The distribution of absolute copy numbers of miRNA-21 and miRNA-141 in six types of cells was obtained. It was found that the number of copies of miRNA-21 and miRNA-141 in different types of cancer cells showed different expression characteristics. The study results can help us more accurately understand cell-to-cell heterogeneity and the relationship between different miRNAs and different types of cancer at the single cell level.


Subject(s)
Biosensing Techniques , MicroRNAs , Neoplasms , Humans , MicroRNAs/analysis , MicroRNAs/genetics , Single-Cell Analysis
10.
Adv Healthc Mater ; 11(12): e2102759, 2022 06.
Article in English | MEDLINE | ID: mdl-35170255

ABSTRACT

Tumor microenvironment (TME)-activated cancer imaging and therapy is a key to achieving accurate diagnosis and treatment of cancer and reducing the side effects. Herein, smart near-infrared carbon dot-metal organic framework MIL-100 (Fe) assemblies are constructed to achieve TME-activated cancer imaging and chemodynamic-photothermal combined therapy. First, a near-infrared emission carbon dot (RCDs) is developed using glutathione (GSH) as the precursor. Then, the RCDs@MIL-100 self-assemblies are obtained using RCDs, FeCl3 , and trimesic acid solutions as raw materials. After the RCDs@MIL-100 enters the TME, a high concentration of GSH reduces Fe3+ to Fe2+ and drains the GSH, triggering the collapse of RCDs@MIL-100 skeleton and the release of RCDs and Fe2+ , at which time the RCDs fluorescence is restored and in an "on" state to illuminate the tumor cells, which achieved cancer imaging. The released Fe2+ reacts with H2 O2 in the TME to form highly reactive hydroxyl radicals (•OH) by Fenton reaction, which achieves the chemodynamic therapy of tumors. Thus, efficient synergistic chemodynamic-photothermal dual mode therapy is achieved under fluorescence imaging guidance with TME response.


Subject(s)
Metal-Organic Frameworks , Nanoparticles , Neoplasms , Carbon , Cell Line, Tumor , Glutathione , Humans , Hydrogen Peroxide , Metal-Organic Frameworks/therapeutic use , Nanoparticles/therapeutic use , Neoplasms/diagnostic imaging , Neoplasms/drug therapy , Tumor Microenvironment
11.
Spectrochim Acta A Mol Biomol Spectrosc ; 269: 120759, 2022 Mar 15.
Article in English | MEDLINE | ID: mdl-34968836

ABSTRACT

It is highly desirable to develop a simple, efficient and sensitive strategy for organophosphorus pesticides (OPs) in both environment pollution and human health. Herein, a novel amplified fluorescence polarization (FP) biosensor was established for highly sensitive detection of OPs using MnO2 nanosheets as the signal enhancer. In this system, OPs can suppress the activity of acetylcholinesterase (AChE) efficiently, blocking the hydrolysis reaction of acetylthiocholine (ATCh) to generate thiocholine (TCh) by AChE. TCh can lead the decomposition of MnO2 nanosheets to manganese ions. So, without the influence of TCh, MnO2 nanosheets can maintain its original shape and form a stable complex with FAM-DNA, which greatly enhanced the FP signal. This method can tremendously improve the sensitivity of FP with a detection limit of 0.01 ng/mL for diazinon. In addition, it was also applicable to determine other four OPs and investigate the level of diazinon in real water samples. Consequently, the proposed approach provides a new promising platform for detection of OPs and is expected to be used in application of environmental monitoring.


Subject(s)
Organophosphorus Compounds/analysis , Pesticides , Acetylcholinesterase , Fluorescence Polarization , Manganese Compounds , Nanostructures , Oxides , Pesticides/analysis
12.
Adv Healthc Mater ; 11(3): e2102073, 2022 02.
Article in English | MEDLINE | ID: mdl-34731532

ABSTRACT

The accurate diagnosis and targeted therapy of malignant tumors face significant challenges. To address these, an oxidized molybdenum polyoxometalate-copper nanocomposite (Ox-POM@Cu) is designed and synthesized here. The doping with Cu determines the formation of oxygen vacancies, which can increase the carrier concentration in Ox-POM@Cu, accelerate electron transfer, and enhance the redox activity, thus playing an efficient catalytic role. The nanocomposite presents unique enzymatic functions characterized by a multielement catalytic activity in the tumor microenvironment (TME). In addition, it can be employed as an NIR-II photoacoustic imaging (PAI) probe and cancer therapy agent. First, it participates in a redox reaction with glutathione (GSH) in tumor tissues, activates the PAI and photothermal therapy functions via NIR-II irradiation, and depletes the GSH supply in cancerous cells. Subsequently, it catalyzes a Fenton-like reaction with H2 O2 in tumor tissues to form hydroxyl radicals, thereby performing a chemodynamic therapy function. The findings show that the developed nanoenzyme is very efficient in the diagnosis and treatment of malignant tumors. This work not only provides a new strategy for the design of TME-induced NIR-II PAI but also presents new insights into enhanced cancer therapy.


Subject(s)
Nanoparticles , Photoacoustic Techniques , Anions , Cell Line, Tumor , Hydrogen Peroxide , Photoacoustic Techniques/methods , Polyelectrolytes , Tumor Microenvironment
13.
Chem Commun (Camb) ; 57(84): 11017-11020, 2021 Oct 21.
Article in English | MEDLINE | ID: mdl-34605511

ABSTRACT

A DNAzyme-mediated target-initiated rolling circle signal amplification strategy based on a microchip platform was developed for detecting apurinic/apyrimidine endonuclease 1 (APE1) at the single-cell level. This strategy was applied to assays of lysate samples from HL-7702, HeLa and MCF-7 cells, with a detection limit of lower than 1 HeLa cell.


Subject(s)
Endonucleases/metabolism , Microarray Analysis/methods , Nucleic Acid Amplification Techniques/methods , Biosensing Techniques , DNA, Catalytic , Electrophoresis , HeLa Cells , Humans , Limit of Detection , MCF-7 Cells , Single-Cell Analysis
14.
ACS Appl Mater Interfaces ; 13(30): 35365-35375, 2021 Aug 04.
Article in English | MEDLINE | ID: mdl-34286953

ABSTRACT

Phototherapy exhibits significant potential as a novel tumor treatment method, and the development of highly active photosensitizers and photothermal agents has drawn considerable attention. In this work, S and N atom co-doped carbon dots (S,N-CDs) with an absorption redshift effect were prepared by hydrothermal synthesis with lysine, o-phenylenediamine, and sulfuric acid as raw materials. The near-infrared (NIR) absorption features of the S,N-CDs resulted in two-photon (TP) emission, which has been used in TP fluorescence imaging of lysosomes and tumor tissue pH and real-time monitoring of apoptosis during tumor phototherapy, respectively. The obtained heteroatom co-doped CDs can be used not only as an NIR imaging probe but also as an effective photodynamic therapy/photothermal therapy (PDT/PTT) therapeutic agent. The efficiencies of different heteroatom-doped CDs in tumor treatment were compared. It was found that the S,N-CDs showed higher therapeutic efficiency than N-doped CDs, the efficiency of producing 1O2 was 27%, and the photothermal conversion efficiency reached 34.4%. The study provides new insight into the synthesis of carbon-based nanodrugs for synergistic phototherapy and accurate diagnosis of tumors.


Subject(s)
Antineoplastic Agents/therapeutic use , Fluorescent Dyes/therapeutic use , Neoplasms/diagnostic imaging , Neoplasms/drug therapy , Photosensitizing Agents/therapeutic use , Quantum Dots/therapeutic use , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/radiation effects , Apoptosis/drug effects , Carbon/chemistry , Carbon/radiation effects , Fluorescent Dyes/chemistry , Fluorescent Dyes/radiation effects , Fluorometry , HeLa Cells , Humans , Hydrogen-Ion Concentration , Lysosomes/metabolism , Mice, Nude , Neoplasms/metabolism , Nitrogen/chemistry , Nitrogen/radiation effects , Photons , Photosensitizing Agents/chemistry , Photosensitizing Agents/radiation effects , Phototherapy , Quantum Dots/chemistry , Quantum Dots/radiation effects , Singlet Oxygen/metabolism , Sulfur/chemistry , Sulfur/radiation effects
15.
Anal Chem ; 93(26): 9218-9225, 2021 07 06.
Article in English | MEDLINE | ID: mdl-34128642

ABSTRACT

The absolute quantification of miRNAs in a single cell allows to better understand the heterogeneity of cells and the relationship between miRNAs and diseases. However, seldom methods for miRNA quantification in a single cell have been reported because the miRNA content in a single cell is very low. Herein, an ultrasensitive chemiluminescence assay strategy based on rolling circle amplification (RCA) on a microchip platform was proposed for the absolute quantification of miRNAs in a single cell. In this strategy, a ring probe with specificity was designed and synthesized, which could perform RCA for target miRNAs to improve the sensitivity and satisfy the need of absolute quantification of miRNAs in a single cell. The 20 liver cancer cells (HepG2) and 20 normal liver cells (HL-7702) were analyzed using this method; it is found that the miRNA-21 contents varied among cells, and miRNA-21 was overexpressed in HepG2 cells. Compared with traditional methods, the proposed strategy has many advantages such as low cost, simple operation, short analysis time, good specificity, and lower probability of false positives. This method is expected to be one of the powerful tools for the absolute quantification of miRNAs in a single cell.


Subject(s)
MicroRNAs , Hep G2 Cells , Humans , Limit of Detection , Luminescence , MicroRNAs/genetics , Nucleic Acid Amplification Techniques , Single-Cell Analysis
16.
Sci Adv ; 7(12)2021 03.
Article in English | MEDLINE | ID: mdl-33741594

ABSTRACT

Tumor microenvironment-responsive therapy has enormous application potential in the diagnosis and treatment of cancer. The glutathione (GSH) level has been shown to be significantly increased in tumor tissues. Thus, GSH can be used as an effective endogenous molecule for diagnosis and tumor microenvironment-activated therapy. In this study, we prepared a tumor microenvironment-induced, absorption spectrum red-shifted, iron-copper co-doped polyaniline nanoparticle (Fe-Cu@PANI). The Cu(II) in this nanoparticle can undergo a redox reaction with GSH in tumors. The redox reaction induces a red shift in the absorption spectrum of the Fe-Cu@PANI nanoparticles from the visible to the near-infrared region accompanying with the etching of this nanoparticle, which simultaneously activates tumor photoacoustic imaging and photothermal therapy, thereby improving the accuracy of in vivo tumor imaging and the efficiency of photothermal therapy. The nanoparticle prepared in this study has broad application prospects in the diagnosis and treatment of cancer.


Subject(s)
Nanoparticles , Neoplasms , Photoacoustic Techniques , Cell Line, Tumor , Humans , Nanoparticles/therapeutic use , Neoplasms/diagnostic imaging , Neoplasms/therapy , Photoacoustic Techniques/methods , Phototherapy , Photothermal Therapy , Polymers , Tumor Microenvironment
17.
Chem Commun (Camb) ; 57(25): 3095-3098, 2021 Mar 28.
Article in English | MEDLINE | ID: mdl-33625437

ABSTRACT

An ultrasensitive chemiluminescence strategy based on signal amplification with a microchip platform was proposed to detect telomerase. This strategy was successfully applied to the determination of lysate samples from HL-7702, HeLa, A549 and MCF-7 cell lines with the detection limit lower than 1 HeLa cell.


Subject(s)
Lab-On-A-Chip Devices , Limit of Detection , Luminescence , Single-Cell Analysis/instrumentation , Telomerase/metabolism , Cell Line, Tumor , Humans
18.
Chem Commun (Camb) ; 56(48): 6579-6582, 2020 Jun 16.
Article in English | MEDLINE | ID: mdl-32400773

ABSTRACT

An intracellular signal amplification strategy was developed for the quantification of ATP in single cells by microchip electrophoresis with laser-induced fluorescence detection. By using the method proposed, intracellular ATP levels in single HeLa, HepG2 and HL-7702 cells were found to be in the range of 30-150, 30-140, and 19-120 fmol per cell, respectively.


Subject(s)
Adenosine Triphosphate/analysis , Electrophoresis, Microchip/methods , Lasers , Adenosine Triphosphate/metabolism , Aptamers, Nucleotide/chemistry , Aptamers, Nucleotide/metabolism , Cell Line, Tumor , Firefly Luciferin/chemistry , Humans , Liposomes/chemistry , Microscopy, Confocal , Reproducibility of Results , Spectrometry, Fluorescence
19.
Anal Chim Acta ; 1100: 258-266, 2020 Mar 01.
Article in English | MEDLINE | ID: mdl-31987149

ABSTRACT

MicroRNAs (miRNAs) are associated with physiological and pathological processes. They are recognized as biomarkers for diseases diagnosis and treatment evaluation. Herein we propose a simple and cost-effective HPLC method for quantitative assay of target miRNAs with femtomolar sensitivity, single-base discrimination selectivity and low background. The assay is based on an innovative signal-on strategy. In this strategy, polyadenylation of poly(A) polymerase extends an all 'A' sequence at the end of target miRNA, and the substantially increased number of adenine bases are labeled with 2-Chloroacetaldehyde (CAA) to open a signal-on mode and realize a signal amplification. The linearly amplified fluorescence signal is separated from other inference signals and quantified by high performance liquid chromatography with fluorescence detection (HPLC-FD). Combining with affinity magnetic solid phase extraction (MSPE), the method is well suited for analysis of complex biological samples such as serum and cell lysate with nearly zero background fluorescence. Taking miRNA-21 as the model analyte, this absolute quantification method has a limit of detection of 200 fM and a linear calibration curve (R2 = 0.999) in the range from 2.00 pM to 1.00 nM. Using locked nucleic acid (LNA) modified probes rather than ssDNA probes, the assay selectivity is improved. Moreover, analysis of bovine serum and cell lysate samples by using the method is demonstrated. Intracellular content of miRNA-21 is found to be 0.0150 amol/cell in MCF-7 cells with an assay repeatability of 4.0% (RSD, n = 3). The present HPLC quantification of miRNA offers an accurate, reliable, and cost-effective means for quantitative assay of miRNAs occurring in biological samples. Also importantly, it eliminates the need for total RNA isolation for the analysis. It may be useful for more effective diagnosis of diseases and therapeutic evaluation.


Subject(s)
Chromatography, High Pressure Liquid/methods , DNA, Single-Stranded/genetics , MicroRNAs/analysis , Animals , Biological Assay , Calibration , Cattle , Cell Culture Techniques , Humans , Limit of Detection , MCF-7 Cells , MicroRNAs/genetics , Nucleic Acid Amplification Techniques
20.
Analyst ; 145(5): 1783-1788, 2020 Mar 07.
Article in English | MEDLINE | ID: mdl-31942587

ABSTRACT

This work describes a novel method for quantification of miRNAs based on multistage signal amplification (MSA) and liquid chromatography-electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). The multistage signal amplification involves hybridization enrichment of miRNA targets with a DNA probe-magnetic bead conjugate, target recycling amplification with a duplex-specific nuclease, and acid hydrolysis of the reporter molecules producing free nucleobases. Nucleobases thus generated are quantified by LC-ESI-MS/MS with specificity and repeatability. Taking miR-21 as the model target, biological samples such as serum and cell cultures were analyzed by using the present protocol. The analytical results indicate that facile and cost-effective quantifications of miRNA targets can be achieved by using the popular LC-ESI-MS/MS technique, and very importantly, without an isolation of total RNAs from the sample prior to the quantitative assay. The assay for miR-21 detection had a linear calibration curve in the range from 0.2 pM to 0.25 nM with a limit of detection of 60 fM. Analysis of MCF-7 cells treated with toremifene (a potent inhibitor of breast cancer cell growth) revealed that the content of miRNA-21 decreased by ca. 50%, and the decrease was dose-dependent.


Subject(s)
Chromatography, Liquid/methods , MicroRNAs/analysis , Spectrometry, Mass, Electrospray Ionization/methods , Tandem Mass Spectrometry/methods , DNA Probes/chemistry , DNA Probes/genetics , DNA, Single-Stranded/chemistry , DNA, Single-Stranded/genetics , Deoxyribonucleases/chemistry , Humans , Immobilized Nucleic Acids/chemistry , Immobilized Nucleic Acids/genetics , Limit of Detection , MCF-7 Cells , MicroRNAs/genetics , Nucleic Acid Hybridization
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