Lysosomes Initiating and DNAzyme-Assisted Intracellular Signal Amplification Strategy for Quantification of Alpha-Fetoprotein in a Single Cell.

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.

A Framework Nucleic Acid-Mediated Multimodal Tandem Multivariate Signal Amplification Strategy for Simultaneous Absolute Quantification of Three Different MicroRNAs in a Single Cell.

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.

Intracellular Multicomponent Synchronous DNA-Walking Strategy for the Simultaneous Quantification of Tumor-Associated Proteins in a Single Cell.

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.

An ultrasensitive bunge bedstraw herb type DNA machine for absolute quantification of mRNA in single cell.

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.

An ultrasensitive multivariate signal amplification strategy based on microchip platform tailored for simultaneous quantification of multiple microRNAs in single cell.

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.

A DNAzyme-mediated target-initiated rolling circle amplification strategy based on a microchip platform for the detection of apurinic/apyrimidinic endonuclease 1 at the single-cell level.

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.

Absolute Quantification of MicroRNAs in a Single Cell with Chemiluminescence Detection Based on Rolling Circle Amplification on a Microchip Platform.

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.

An ultrasensitive chemiluminescence strategy based on a microchip platform for telomerase detection at a single-cell level.

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.

TGF-ß1 pathway affects the protein expression of many signaling pathways, markers of liver cancer stem cells, cytokeratins, and TERT in liver cancer HepG2 cells.

Liver cancer is one of the most common human malignancies, and transforming growth factor-beta (TGF-ß) pathway plays a key role in its pathogenesis. To study the relationship between TGF-ß pathway and the related protein expression of many signaling pathway, markers of stem cells, CK family, and others, liver cancer HepG2 cells were transfected with siRNA directed against TGF-ß1 or were treated with exogenous TGF-ß1. Then, these protein levels were measured by Western blotting. After siRNA transfection, TGF-ß1 protein level was decreased, indicating that the siRNA against it was effective. In exogenous TGF-ß1 group, the expression of smad4, smad2/3, and ß-catenin proteins was increased, whereas that of p-smad2/3, CD133, cleaved Notch1, and epithelial cell adhesion molecule (EpCAM) proteins at 48 h was decreased. The expression of CK8 and CK18 proteins was increased at 24 h and was decreased at 48 and 96 h. In TGF-ß1-silenced group, the expression of smad2/3, ß-catenin, cleaved-notch1, and CK18 proteins was decreased, while that of smad4, p-smad2/3, CD133, EpCAM, and CK8 proteins was increased. TERT protein expression was slightly increased in exogenous TGF-ß1 group at 48 h and in TGF-ß1-silenced group at 96 h. TGF-ß1 did not affect the protein expression of CK19 and HIF-1. Thus, TGF-ß1 pathway plays an important role in cell regulation of liver cancer through the modulation of these proteins. These data will contribute to the understanding of the pathogenesis of liver cancer and the role of TGF-ß pathway in this process.