Chunyan Chen

Double-Strand Displacement Biosensor and Quencher-Free Fluorescence Strategy for Rapid Detection of MicroRNA.

We describe a facile quencher-free fluorescence strategy for rapid detection of microRNAs (miRNAs) by using a novel double-strand displacement sensor. The sensor is designed with an outstanding 2-aminopurine (2-AP) fluorophore as a probe and a predesigned cDNA, which can completely complement the target miRNA and partly complement the 2-AP probe. When the target miRNA is added, the cDNA can be competed off from the cDNA\2-AP probe duplex, thereby forming a cDNA\RNA heteroduplex. The free 2-AP probe induces an increase in the fluorescent signal. A limit of detection of 5 nM and a wide linear range from 5 to 1000 nM (R(2) = 0.9971) are achieved by this assay. The rapid detection strategy can be accomplished within 2 h without expensive nanoparticles and complicated instruments for the whole procedure, thus, offering a significant potential for clinical application.

Rapid and efficient separation of glycoprotein using pH double-responsive imprinted magnetic microsphere

As biomarkers of many diseases, glycoproteins are of great significance to clinical diagnostics. However, the determination of low abundant glycoproteins in complex biological samples without any pretreatment process is still a problem. In this study, a rapid and convenient separation method for highly efficient enrichment of glycoproteins is reported, based on pH double-responsive imprinted magnetic microspheres. Thin imprinted polymer shells were fabricated onto the surface of magnetic microspheres by free radical polymerization, using 2-(Dimethylamino) ethyl methacrylate as pH-sensitive monomer, 4-vinylphenylbronic acid as boronate affinity monomer, and ovalbumin (OVA) as template molecule. Combining the advantages of pH-sensitive monomer and boronate affinity monomer, rapidly capture-release of OVA could be modulated by changing solution pH. Moreover, high absorption ability (81.2mg/g) was achieved within about 10min. This study provided responsible way to imprint glycoproteins and showed great potential for glycoprotein detection in clinical diagnostic.

A virus resonance light scattering sensor based on mussel-inspired molecularly imprinted polymers for high sensitive and high selective detection of Hepatitis A Virus

We described a novel resonance light scattering (RLS) sensor for the specific recognition of trace quantities of Hepatitis A Virus (HAV); the sensor was based on a mussel-inspired hepatitis molecularly imprinted polymer. As a recognition element, polydopamine (PDA)-coated totivirus-imprinted polymer was introduced on the surface of SiO2 nanoparticles (virus-imprinted SiO2@PDA NPs) using an efficient one-step synthesis method. The target virus was selectively captured by the imprinted polymer films, thereby increasing the RLS intensity. A simple fluorescence spectrophotometer was employed to measure the changes in the intensity. The enhanced RLS intensity (∆IRLS) was proportional to the concentration of HAV in the range of 0.04-6.0nmol∙L-1, with a low limit of detection of 8.6pmol∙L-1. The selectivity study confirmed that the resultant HAV-imprinted SiO2@PDA NPs possessed high selectivity for HAV. The sensor was successfully applied for the direct detection of additional HAV from a 20,000-fold dilution of human serum. The proposed strategy is simple, eco-friendly, highly selective, and sensitive.

A novel CdTe quantum dots probe amplified resonance light scattering signals to detect microRNA-122

We report a rapid and facile resonance light scattering (RLS) technique that utilizes CdTe quantum dots (CdTe QDs) probe to detect microRNA-122. The RLS sensor is ingeniously designed with P1 and P2, two cDNA sequence probes with partially complementary sequences to miRNA-122. The amine-modified P1 and P2 are coupled to the surface of QDs to form functional QDs-P1 and QDs-P2 conjugates, which are collectively referred to as QDs-P. The cDNAs hybridize with the target miRNA to rapidly induce the self-assembly of QDs probe and change RLS intensity. The proposed technique can detect miRNA-122 within 40min. RLS intensity is enhanced in proportion with miRNA-122 concentrations of 0.16-4.80nM and has a low detection limit of 9.4pM. In addition, the assay satisfactorily detects miRNAs in human serum samples. Thus, the assay has considerable potential for the analysis of other interesting tumor makers.

Click on the bidirectional switch: the aptasensor for simultaneous detection of lysozyme and ATP with high sensitivity and high selectivity.

A bifunctional and simple aptasensor was designed to one-spot simultaneously detect two analytes, lysozyme and ATP. The aptasensor was obtained by the electronic interaction between methyl violet (MV) and dsDNA. The dsDNA was obtained by hybridization of ATP aptamer and lysozyme aptamer. And we used the resonance light scattering (RLS) technique to detect the concentration of lysozyme and ATP. During the procedure of detection, the aptasensor works like a bidirectional switch, the corresponding side of the dsDNA will open when the target (lysozyme or ATP) “click” the aptamer, which results in corresponding RLS signal change. By the combination of the RLS technique, it is found that the changed RLS intensity was proportional to the concentration of lysozyme and ATP. The mixtures of ATP and lysozyme also met two binary function relations. The results indicated that the aptasensor could achieve simultaneous detection of ATP and lysozyme, the detection limits of ATP and lysozyme could reach 10−11 M and 10−12 M, respectively. The aptasensor shows potential application for small molecule and protein detection by RLS, it could extend the application of RLS technique.

2-aminopurine probe in combination with catalyzed hairpin assembly signal amplification for simple and sensitive detection of microRNA

A quencher-free and enzyme-free fluorescent sensor was proposed to simply and sensitively detect miRNA via the target catalyzed hairpin assembly (CHA) signal amplification in combination with 2-aminopurine (2-AP) molecular beacon (MBs). This sensor contains two DNA hairpins termed as H1 and H2. H1 labeled by 2-AP needs no quenchers because 2-AP can be quenched through its stacking interaction with the adjacent bases. H2 is partially complementary to H1. In the presence of the target microRNA (miRNA), H1 is unfolded and produces the DNA/RNA complexes, enhancing the fluorescent signal. Then, the RNA of the DNA/RNA complexes can be displaced by H2 and the free miRNA can interact with another H1, resulting in the significant fluorescence enhancement of the system. This signal amplification process is enzyme-free, making the sensor more simple and cost effective. The detection limit of this sensor could be as low as 3.5pM. We further applied this assay to monitor the overexpressed miRNA-21 from human breast cancer cells to confirm its applicability. The proposed sensor could be used as a simple and sensitive platform for target miRNA detection, holding great potential for convenient monitoring of different miRNA biomarkers for early diagnosis of various cancers.

Ratiometric Fluorescence Sensor for the MicroRNA Determination by Catalyzed Hairpin Assembly

A novel catalyzed hairpin assembly-based turn-on ratiometric fluorescence biosensor was constructed for the determination of microRNA-122 (miRNA-122) by using 2-aminopurine (2-AP) and thioflavin T (ThT) as detection signal sources. Hairpin DNA sequence (H1) includes the complementary strands of miRNA-122 and G-quadruplex-forming sequence. When miRNA-122 was presented, hybridization occurred between miRNA-122 and part of H1, causing a double-stranded DNA and a G-quadruplex formed. The formed double-stranded DNA significantly decreased the fluorescence intensity of 2-AP. Furthermore, after binding with ThT, the formed G-quadruplex led to the fluorescent enhancement. The hairpin DNA sequence (H2) hybridized with the unfolded H1 and displaced miRNA-122. Finally, the displaced miRNA-122 again hybridized with the H1 and initiated cycle amplification. This sensor showed a linear ranges of 0.5-50 nM and the limit of detection for miRNA-122 assay was 72 pM (with the lowest measured concentration of 500 pM) for determination of miRNA-122 when no other miRNA was present. Measurements on cell lysates from 100, 1000, and 10 000 cells of three different cell lines provided increasing signal ratios, which showed the application potential of the sensor for miRNA determination in real samples.

Simple G-quadruplex-based 2-aminopurine fluorescence probe for highly sensitive and amplified detection of microRNA-21

Based on 2-aminopurine (2-AP) probe in conjunction with a G-quadruplex structure and signal amplification technique, a simple and highly sensitive fluorescence sensor for detecting microRNA (miRNA) is developed for high signal-to-background ratio and wide linear range. The proposed sensor contains two hairpins DNA: H1 and H2. H1 is labeled by 2-AP incorporated into a G-rich sequence. Upon the addition of a target miRNA, H1 is unfolded and forms DNA/RNA complexes that contain a G-quadruplex, thereby significantly enhancing 2-AP fluorescence due to the protection provided by the G-quadruplex. Subsequently, H2 can displace the miRNA from the DNA/RNA complexes and induce signal amplification, resulting in further enhanced fluorescence intensity. Hence, the sensor is highly sensitive and its low limit of detection (L.O.D.) can reach as low as 1.48pM. Furthermore, the proposed sensor is used to detect overexpressed miRNA-21 from human breast cancer cell lysate. The result demonstrates the potential of the proposed sensor to monitor different miRNA biomarkers for the early diagnosis of various cancers.

Reduced graphene oxide as a resonance light-scattering probe for thrombin detection using dual-aptamer-based dsDNA

This paper describes a reduced graphene oxide (RGO)-based resonance light-scattering (RLS) method for thrombin detection by using double strand DNA (dsDNA) as a binding element. dsDNA is obtained by hybridizing DNA1 and DNA2, which respectively consist of one aptamer of thrombin and the complementary strand of the other aptamer of thrombin. When thrombin is added, the specific binding of two aptamers to thrombin results in a complex (DNA1-thrombin-DNA2) and triggers the release of the complementary strand of two aptamers from dsDNA. The released ssDNA can be self-assembled on the surface of RGO to form a stable DNA1-thrombin-DNA2-RGO complex, which increases RLS signals. This simple and rapid method has enabled the detection of thrombin in the picomolar level in buffer and human serum samples. This study is the first to use RGO as a platform in RLS sensor, which can extend the application of RGO.

A virus-MIPs fluorescent sensor based on FRET for highly sensitive detection of JEV

Major stumbling blocks in the recognition and detection of virus are the unstable biological recognition element or the complex detection means. Here a fluorescent sensor based on virus-molecular imprinted polymers (virus-MIPs) was designed for specific recognition and highly sensitive detection of Japanese encephalitis virus (JEV). The virus-MIPs were anchored on the surface of silica microspheres modified by fluorescent dye, pyrene-1-carboxaldehyde (PC). The fluorescence intensity of PC can be enhanced by the principle of fluorescence resonance energy transfer (FRET), where virus acted as energy donor and PC acted as energy acceptor. The enhanced fluorescence intensity was proportional to the concentration of virus in the range of 24-960pM, with a limit of detection (LOD, 3σ) of 9.6pM, and the relative standard deviation was 1.99%. In additional, the specificity study confirmed the resultant MIPs has high-selectivity for JEV. This sensor would become a new key for the detection of virus because of its high sensitive, simple operation, high stability and low cost.