MicroRNAs (miRNAs) participate in the significant processes of life course, can be used as quantificational biomarkers for cellular level researches and related diseases. Conventional methods of miRNAs’ quantitative detection are obsessed with low sensitivity, time and labour consuming. Otherwise, the emerging miRNAs detection approaches are mostly exposed to the expensive equipment demands and the professional operation, remains at the stage of laboratory and concept demonstration phase. Herein, we designed a novel miRNAs detection platform that based on enzyme-free DNA circuits and electrochemical luminescence (ECL). MicroRNA21 was chosen as the ideal analyte of this platform. The whole process consists of enzyme-free DNA circuits and ECL signal giving-out steps, achieves advantages of operating in constant temperature condition, without the participation of the enzyme, preferable sensitivity and specificity. Through this approach, the sensitivity achieved at 10pM. It is indicated that this miRNAs detection platform possesses potentials to be an innovation of miRNA detection technologies in routine tests. Benefits of the high penetration of ECL in well-equipped medical establishment, this approach could greatly lessen the obstacles in process of popularization and possess excellent prospects of practical application.
Food-borne pathogens such as Listeria monocytogenes have been recognized as a major cause of human infections worldwide, leading to substantial health problems. Food-borne pathogen identification needs to be simpler, cheaper and more reliable than the current traditional methods. Here, we have constructed a low-cost paper biosensor for the detection of viable pathogenic bacteria with the naked eye. In this study, an effective isothermal amplification method was used to amplify the hlyA mRNA gene, a specific RNA marker in Listeria monocytogenes. The amplification products were applied to the paper biosensor to perform a visual test, in which endpoint detection was performed using sandwich hybridization assays. When the RNA products migrated along the paper biosensor by capillary action, the gold nanoparticles accumulated at the designated Test line and Control line. Under optimized experimental conditions, as little as 0.5 pg/μL genomic RNA from Listeria monocytogenes could be detected. The whole assay process, including RNA extraction, amplification, and visualization, can be completed within several hours. The developed method is suitable for point-of-care applications to detect food-borne pathogens, as it can effectively overcome the false-positive results caused by amplifying nonviable Listeria monocytogenes.
MicroRNAs (miRNAs) play pivotal roles in many fundamental aspects of life, such as tissue differentiation, metabolic
modulation and cell proliferation control. As miRNA has the characteristics of small size, easy degradation and low
abundance, a simple, rapid and specificity method to measure the concentration of miRNA is required. Herein, we
present an Electrochemiluminescent Chips system for rapid miRNA detection based on the base stacking hybridization
and magnetic beads (MB) enrichment technology. In this design, integrating the microfluidic system with
Electrochemiluminescent detection makes it easy to assemble the multiple assay steps (e.g., sample preparation,
incubation, washing and detection) and let the device convenient to carry. This method is very fast, we can detect
miRNA in 19 min with a 10 uL sample volume. Moreover, in this experiment the detection limit was 1 fmol of the pure
synthetic miRNA, and the linear range was from 5 fmol to 1 pmol. It also exhibited excellent selectivity over a series of
interference miRNA, which display a high level of expression in cancer cell. These results suggest that this method
might have potential for cancer diagnosis at the point of care.
Rotaviruses are double-stranded RNA viruses belonging to the family of enteric pathogens. It is a major cause of
diarrhoeal disease in infants and young children worldwide. Consequently, rapid and accurate detection of rotaviruses is
of great importance in controlling and preventing food- and waterborne diseases and outbreaks. Reverse
transcription-polymerase chain reaction (RT-PCR) is a reliable method that possesses high specificity and sensitivity. It
has been widely used to detection of viruses. Electrochemiluminescence (ECL) can be considered as an important and
powerful tool in analytical and clinical application with high sensitivity, excellent specificity, and low cost. Here we have
developed a method for the detection of rotavirus by combining in situ magnetic beads (MBs) based RT-PCR with ECL.
RT of rotavirus RNA was carried out in a traditional way and the resulting cDNA was directly amplified on MBs.
Forward primers were covalently bounded to MBs and reverse primers were labeled with tris-(2, 2’-bipyridyl) ruthenium
(TBR). During the PCR cycling, the TBR labeled products were directly loaded and enriched on the surface of MBs.
Then the MBs–TBR complexes could be analyzed by a magnetic ECL platform without any post-modification or
post-incubation,which avoid some laborious manual operations and achieve rapid yet sensitive detection. In this study,
rotavirus from fecal specimens was successfully detected within 2 h, and the limit of detection was estimated to be
104copies/μL. This novel in situ MBs based RT-PCR with ECL detection method can be used for pathogen detection in
food safety field and clinical diagnosis.
Methylase is vital for a large number of biological reactions. Here we developed a new method for DNA methylase
activity analysis. In this paper, a DNA hairpin probe with a sequence of 5'-GATC-3' in the stem region was designed.
The 5'-GATC-3' sequence was targeted by Dam MTase and was methylated. Subsequently, restriction enzyme Dpnl recognized the site and cut it. Then the haipin probe was transformed into three single stranded DNA. This enzymatic
process can be monitored by the change of SYBR green I fluorescence. The current label free assay is an useful tool for
DNA methylase activity analysis due to its simplicity, speedability, and low cost.
Recently, our group showed that one can detect specific oligonucleotides at low femtomolar levels with the
electrochemiluminescence (ECL) biobarcode approach based on tris-(2, 2'-bipyridyl) ruthenium (TBR)-labeled
cysteamine. It would be a significant advance to use the cysteamine assisted ECL biobarcode assay to detect protein
targets in addition to DNA targets. Taking advantage of sandwich binding of two affinity aptamers for increased
specificity, TBR-cysteamine as biobarcode for signal amplification and magnetic beads based ECL technology for rapid
detection, a promising assay for thrombin quantification is developed. The sandwich complex could be selectively
captured by micromagnetic particles and then quantified by ECL signals. Current cysteamine-Gold nanoparticle (GNP)
conjugates based ECL biobarcode assay is expected to become a powerful tool for protein analysis.
The polycomb group protein enhancer of zeste homolog 2 (EZH2) regulating cell cycle and functioning
as a transcriptional repressor, is overexpressed in several human cancers. Therefore it can be a
molecular marker for detection of cancer progression and metastasis. Here the
electrochemiluminescence (ECL) assay was developed to detect and quantify the amount of EZH2
mRNA expression in cancer cell. Total mRNA was reverse transcribed into cDNA. The cDNA was
amplified using a forward and reverse primer pair which were labeled with biotin and Tris (2,
2-bipyridine) ruthenium (II) (TBR) on the 5' end, respectively. The amplification product was captured
on streptavidin coated magnetic beads and then separated using a magnetic field. The TBR labels were
reacted with the most efficient coreactant, TPA, on the electrode. Photons were produced and detected
by a custom-built ECL system. The housekeeping gene hydroxymethylbilane synthase (HMBS) was
used as an approximate reference to quantify the amount of EZH2 mRNA expression, whose primer
pairs were labeled the same as EZH2. Result indicated that the EZH2 mRNA was overexpressed in
MCF-7 cells relative to normal blood cells. This assay is specific and sensitive and could be used for
the clinical diagnosis and prognosis of cancer.
The technology of single-base mutation detection plays an increasingly important role in diagnosis and prognosis of
genetic-based diseases. Here we reported a new method for the analysis of point mutations in genomic DNA through the
integration of allele-specific oligonucleotide ligation assay (OLA) with magnetic beads-based electrochemiluminescence
(ECL) detection scheme. In this assay the tris(bipyridine) ruthenium (TBR) labeled probe and the biotinylated probe are
designed to perfectly complementary to the mutant target, thus a ligation can be generated between those two probes by
Taq DNA Ligase in the presence of mutant target. If there is an allele mismatch, the ligation does not take place. The
ligation products are then captured onto streptavidin-coated paramagnetic beads, and detected by measuring the ECL
signal of the TBR label. Results showed that the new method held a low detection limit down to 10 fmol and was
successfully applied in the identification of point mutations from ASTC-α-1, PANC-1 and normal cell lines in codon 273
of TP53 oncogene. In summary, this method provides a sensitive, cost-effective and easy operation approach for point
mutation detection.
Telomerase, a ribonucleoprotein enzyme that adds telomeric repeats to the 3'end of chromosomal DNA for
maintaining chromosomal integrity and stability. This strong association of telomerase activity with tumors
establishing it is the most widespread cancer marker. A number of assays based on the polymerase chain
reaction (PCR) have been developed for the evaluation of telomerase activity. However, those methods require
gel electrophoresis and some staining procedures. We developed an electrochemiluminescent (ECL) sensor for
the measuring of telomerase activity to overcome these problems such as troublesome post-PCR procedures and
semi-quantitative assessment in the conventional method. In this assay 5'-biotinylated telomerase synthesis (TS)
primer serve as the substrate for the extension of telomeric repeats under telomerase. The extension products
were amplified with this TS primer and a tris-(2'2'-bipyridyl) ruthenium (TBR)-labeled reversed primer. The
amplified products was separated and enriched in the surface of electrode by streptavidin-coated magnetic beads,
and detected by measuring the ECL signals of the TBR labeled. Measuring telomerase activity use the sensor is
easy, sensitive, rapid, and applicable to quantitative analysis, should be clinically useful for the detection and
monitoring of telomerase activity.
It is well known that plant and animal viruses had widely spread the whole of world, and made a big loss in farming and
husbandry. It is necessary that a highly efficient and accurate virus's detection method was developed. This research
combines reverse transcription polymerase chain reaction (RT-PCR) technique with electrochemiluminescence method,
to detect plant RNA viruses for the first time. Biotin-probe hybridizes with PCR product to specific select the target for
detection, thus can avoid pseudo-positive result. TBR-probe hybridizes with PCR product to emit light for ECL detection.
Specific nucleic acid sequences (20bp) were added to 5' terminal all of the primers, which can improve the chance of
hybridization between TBR-probe and PCR product. At the same time, one of the PCR product chain can hybridize two
Ru-probes, the ECL signal is intensified. The method was used to detect Odntoglossum ringspot virus ORSV, Sugarcane
mosaic virus ScMV, Sorghum mosaic virus SrMV, and Maize dwarf mosaic virus MDMV, the experiment results show
that this method could reliably identity virus infected plant samples. In a word, this method has higher sensitivity and
lower cost than others. It can effectively detect the plant viruses with simplicity, stability, and high sensitivity.
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