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In vitro detection of low abundance biomolecules including microRNAs (miRNAs) is essential to biological research and early clinical diagnosis. In this work, a versatile magnetic bead (MB)-based flow cytometric assay was developed for the detection of hsa-miR-221-3p, which is strongly associated with papillary thyroid carcinoma (PTC). In the presence of hsa-miR-221-3p, the complementary DNA probe attached to the surface of MBs is hybridized with the target to form DNA/RNA heteroduplexes. After the recognition of the DNA/RNA heteroduplexes by PicoGreen, the fluorescence signals of each MB were readily detected using a flow cytometer. This assay can selectively detect hsa-miR-221-3p with a detection limit of 2.1 pM. The practicality of the assay is demonstrated by the discrimination of thyroid cancer tissues from normal tissues, and a satisfactory result is obtained. Moreover, this assay can be rapidly carried out in one step at room temperature, providing a generic method for the sensitive detection of miRNAs in molecular diagnosis.

A facile method has been developed for the rapid and efficient enrichment of DNAs from different media including synthetic single-strand DNAs (ssDNAs) from buffer solutions and cell-free DNAs (cfDNAs) from blood plasma through electric field-driven adsorption and desorption of DNAs by a polyacrylamide/phytic acid/polydopamine (PAAM/PA/PDA) hydrogel. The as-prepared PAAM/PA/PDA hydrogel possesses regular porosity with a large surface area, strong electric field responsiveness/good conductivity, and a rich aromatic structure, which can be used as an ideal adsorbent for DNA enrichment under a positive electric field. The enriched DNAs can be released efficiently when the positive electric field is converted to a negative electric field. The PAAM/PA/PDA hydrogel-based electrochemical method enables the completion of the process of DNA adsorption and release within 5 min and exhibits reasonable enrichment efficiencies and recovery rates of various DNAs. For instance, the high enrichment sensitivity (0.1 pmol L-1) together with the excellent recovery (>75%) of an ssDNA with 78 nucleotides is obtained. Combined with the PCR amplification technique, the practicability of the as-proposed method is demonstrated by the screening of circulating tumor DNAs (ctDNAs) with a BRAFV600E mutation in cfDNAs from the blood plasma samples of patients with papillary thyroid cancer or thyroid nodule and random patients from a clinical laboratory.

In this work, a simple enzyme-free flow cytometric assay (termed as TSDR-based flow cytometric assay) has been developed for the detection of papillary thyroid carcinoma (PTC)-related microRNA (miRNA), hsa-miR-146b-5p with high performance through the toehold-mediated strand displacement reaction (TSDR) on magnetic beads (MBs). The complementary single-stranded DNA (ssDNA) probe of hsa-miR-146b-5p was first immobilized on the surface of MB, which can partly hybridize with the carboxy-fluorescein (FAM)-modified ssDNA, resulting in strong fluorescence emission. In the presence of hsa-miR-146b-5p, the TSDR is trigged, and the FAM-modified ssDNA is released form the MB surface due to the formation of DNA/RNA heteroduplexes on the MB surface. The fluorescence emission change of MBs can be easily read by flow cytometry and is strongly dependent on the concentration of hsa-miR-146b-5p. Under optimal conditions, the TSDR-based flow cytometric assay exhibits good specificity, a wide linear range from 5 to 5000 pM and a relatively low detection limit (LOD, 3σ) of 4.21 pM. Moreover, the practicability of the assay was demonstrated by the analysis of hsa-miR-146b-5p amounts in different PTC cells and clinical PTC tissues.

Accurate and sensitive detection of disease-related microRNAs (miRNAs) is of great significance for early disease diagnosis. In this work, a DNA tetrahedron nanoprobe (DTNP)-based fluorescence resonance energy transfer (FRET) sensing platform (termed DTNP sensor) was constructed for sensitive detection of tumor-related miRNA (e.g., hsa-miR-146b-5p) with DNA assisted cyclic amplification. DTNP was synthesized by DNA self-assembly. In the absence of hsa-miR-146b-5p, the fluorescence DNA (HP) modified with FAM at the 5' terminal and TAMRA at the 3' terminal cannot form the hairpin structure because of the hybridization with the extended DNA strand of the DNA tetrahedron, resulting in a low FRET effect. In the presence of hsa-miR-146b-5p, it would complementarily hybridize with the extended DNA strand of the DNA tetrahedron, leading to the release of HP and occurrence of strong FRET. Thus, the concentration of hsa-miR-146b-5p can be revealed by the change in the fluorescence intensity. Moreover, an assistant DNA was employed to replace hsa-miR-146b-5p for cyclic signal amplification, which can further enhance the detection sensitivity. Under the optimal experimental conditions, the limit of detection for hsa-miR-146b-5p was as low as 6 pM (S/N = 3). Furthermore, the DTNP sensor was successfully applied to evaluate the hsa-miR-146b-5p expression levels in different cell lines. The inhibition of hsa-miR-146b-5p expression in different cells was also investigated and a satisfactory result was obtained.

We developed a three-dimensional (3D) polymer-brush substrate for protein and peptide microarray fabrication, and this substrate was facilely prepared by copolymerization of glycidyl methacrylate (GMA) and 2-hydroxyethyl methacrylate (HEMA) monomers via surface-initiated atom transfer radical polymerization (SI-ATRP) on a glass slide. The performance of obtained poly(glycidyl methacrylate-co-2-hydroxyethyl methacrylate) (P(GMA-HEMA)) brush substrate was assessed by binding of human IgG with rabbit antihuman IgG antibodies on a protein microarray and by the determination of matrix metalloproteinase (MMP) activities on a peptide microarray. The P(GMA-HEMA) brush substrate exhibited higher immobilization capacities for proteins and peptides than those of a two-dimensional (2D) planar epoxy slide. Furthermore, the sensitivity of the P(GMA-HEMA) brush-based microarray on rabbit antihuman IgG antibody detection was much higher than that of its 2D counterpart. The enzyme activities of MMPs were determined specifically with a low detection limit of 6.0 pg mL(-1) for MMP-2 and 5.7 pg mL(-1) for MMP-9. By taking advantage of the biocompatibility of PHEMA, the P(GMA-HEMA) brush-based peptide microarray was also employed to evaluate the secretion of MMP-2 and MMP-9 by cells cultured off the chip or directly on the chip, and satisfactory results were obtained.

Matrix metalloproteinases (MMPs) are believed to play an important role in tumor invasion. Herein, a peptide microarray-based fluorescence assay has been proposed for simultaneously determining the activities of MMP-2 and MMP-9 through the strong binding affinity of fluorescein isothiocyanate modified avidin (avidin-FITC) with the immobilized biotinylated peptide substrate on the microarray. In the presence of MMPs, the biotin moiety is released from the microarray by enzymatic cleavage of the peptide substrate, resulting in a significant decrease of the fluorescence signal. Under the optimal experimental conditions, the fluorescence intensity changes (ΔF%) are proportional to the concentrations of MMP-2 and MMP-9 within the ranges of 50 pg mL−1 to 50 ng mL−1 and 50 pg mL−1 to 100 ng mL−1 in the enzyme mixture, respectively. The detection limits are 45 pg mL−1 for MMP-2, and 60 pg mL−1 for MMP-9. In particular, the activities of extracellular MMP-2 and MMP-9 are determined by the peptide microarray-based fluorescence assay, and satisfactory results are obtained.

Matrix metalloproteinases (MMPs) are important biomarkers of various tumors. Herein, a peptide microarray-based fluorescence assay is developed for quantitatively profiling of MMP-2 activity in different matrices through the binding of the immobilized biotinylated peptides on the microarray with the fluorescein isothiocyanate (FITC) modified neutravidins. In the presence of MMP-2, the biotin moiety is released from microarray by enzymatic cleavage of peptide substrate, resulting in the decrease of fluorescence signal. The change of fluorescence intensity is correlated with MMP-2 activity. The detection limit down to 14 pg mL−1 in buffer solution is obtained by a selected peptide substrate for MMP-2 from eleven peptide substrate candidates. The cellular secreted MMP-2 activity levels of different living cells are further successfully quantitatively evaluated by the selected peptide substrate. Using mouse-bearing MG-63 human osteosarcoma as a model system, we also demonstrate that the activity of MMP-2 in serum is closely related with the cancer progression.

Menopause is characterized by abnormal oestrogen levels. In our preliminary research, we identified that Cistanche deserticola exerts oestrogenic effects, and hypothesized it to be because of its glycoside constituents. In this study, we orally administered C. deserticola total glycosides (CDTG) daily at a dose of 1.5 g/kg body weight to sexually immature female Kunming mice. The negative and positive control groups received equal volumes of distilled water and diethylstilboestrol solution, respectively. On the fifth day, blood samples were collected, and the uteri were immediately excised and weighed. Proliferation of MCF7 cells was evaluated after they were exposed to the serum from each group of CDTG-treated mice. We used cluster analysis to distinguish the CDTG samples collected from 10 different sites. Further, 43 glycosides were identified in CDTG by UPLC/Q-TOF-MS. Finally, principal component, Greyrelativity, and bivariate correlation analysis were used to identify eight components that contributed to the oestrogenic activity of CDTG; they were sinapaldehyde glucoside, salidroside, syringin, dehydrodiconiferyl alcohol-4-O-β-D-glucoside, echinacoside, ononin, bartsioside, and kankanoside I. Among them, sinapaldehyde glucoside, syringin, dehydrodiconiferyl alcohol-4-O-β-D-glucoside, bartsioside and kankanoside I are first reported as oestrogenic active glycosides. Further studies are required to investigate the in vivo activity and safety of the identified components.

Highly accurate analysis of single-nucleotide polymorphisms (SNPs) plays an important role in both disease diagnostics and personalized medicine development. In this work, a DNA microarray-based resonance light scattering (RLS) assay has been developed for multiplexed detection of papillary thyroid carcinoma (PTC) related mutation points including BRAFV600E (t1m), NRAS codon 61 (t2m), TERT promoter g.1295228 (t31m) and TERT promoter g.1295250 (t32m) with high sensitivity and selectivity by the attachment of polyvalent ssDNA modified 13 nm gold nanoparticles (ssDNAs@GNPs) followed by silver deposition for signal enhancement. The microarray-based RLS assay provides a detection limit (S/N = 3) at the sub-nanomolar level for the target ssDNAs and determines allele frequencies as low as 0.2% for t1m, 0.2% for t2m, 0.5% for t31m, and 0.5% for t32m in the cocktail of target ssDNAs, respectively. The practicability of the DNA microarray-based RLS assay is demonstrated by profiling of t2m in 50 clinical thyroid tissue samples of PTC patients, and satisfactory results are obtained.

Fisher discrimination dictionary sparse learning (FDDL) has led to interesting image recognition results where the Fisher discrimination criterion is subject to the coding coefficients. But Fisher discrimination criterion has the limitations of data distribution assumptions and does not consider the local manifold structure of the coding coefficients. In this paper, we will introduce a novel Fisher discrimination sparse learning based on graph embedding (GE-FDSL) scheme. First, we utilizes graph embedding framework to define intra-class compact matrix and inter-class separable matrix imposed on the coding coefficients of training samples to preserving the intra-class compactness and the inter-class separability for the training samples, which simultaneously consider the local manifold structure and label information of the coding coefficients. Then, a new Fisher discrimination criterion based on graph embedding is added to the object function of the sparse coding problem so that the coding coefficients have more discriminative power, where the dictionary atoms in the sparse coding model are associated with the class labels so that the reconstructed error is applied to classification. This method can learn a structured dictionary and sparse coefficients, and in the meantime, it will also keep the local manifold structure of the coding coefficients. So, they will be more discriminative. Experiments on many image databases show that the our algorithm has good classification and recognition performance.

The mapping of specific single nucleotide polymorphisms (SNPs) in patients’ genome is a critical process for the development of personalized therapy. In this work, a DNA microarray-based resonance light scattering (RLS) assay has been developed for multiplexed detection of breast cancer related SNPs with high sensitivity and selectivity. After hybridization of the desired target single-stranded DNAs (ssDNAs) with the ssDNA probes on a microarray, the polyvalent ssDNA modified 13 nm gold nanoparticles (GNPs) are employed to label the hybridization reaction through the formation of a three-stranded DNA system. The H2O2-mediated enlargement of GNPs is then used to enhance the RLS signal. The microarray-based RLS assay provides a detection limit of 10 pM (S/N = 3) for the target ssDNA and determines an allele frequency as low as 1.0% in the target ssDNA cocktail. Combined with an asymmetric PCR technique, the proposed assay shows good accuracy and sensitivity in profiling 4 SNPs related to breast cancer of three selected cell lines.

Recently, the projective dictionary pair learning (DPL) method has obtained better classification representation accuracy which learned a synthesis dictionary and an analysis dictionary to achieve the goal of signal representation. In order to obtain more discriminative ability of the dictionary pair, a new method based on DPL, called discrimination projective dictionary pair learning based dictionary learning method (DPDPL), will be proposed. In DPDPL, we will consider both the inter-class and intra-class incoherence constraints of the synthesis dictionary, and the analysis dictionary should be used to simultaneously maximize the total scatter and the between-class scatter of the signal after coding. Thus, the method ensures that the dictionary has better discriminative ability and the signals are more separable after coding. Experiments of sparse representation-based classification on several face databases show the good performances of the proposed dictionary learning method.