Chip-based visual detection of microRNA using DNA-functionalized gold nanoparticles

In the present study, we developed a highly sensitive and convenient biosensor consisting of gold nanoparticle(Au NP) probes and a gene chip to detect micro RNAs(mi RNAs). Specific oligonucleotides were attached to the glass surface as capture probes for the target mi RNAs, which were then detected via hybridization to the Au NP probes. The signal was amplified via the reduction of HAu Cl4 by H2O2. The use of a single Au NP probe detected 10 pmol L?1 of target mi RNA. The recovery rate for mi R-126 from fetal bovine serum was 81.5%–109.1%. The biosensor detection of mi R-126 in total RNA extracted from lung cancer tissues was consistent with the quantitative PCR(q PCR) results. The use of two Au NP probes further improved the detection sensitivity such that even 1 fmol L?1 of target mi R-125a-5p was detectable. This assay takes less than 1 h to complete and the results can be observed by the naked eye. The platform simultaneously detected lung cancer related mi R-126 and mi R-125a-5p. Therefore, this low cost, rapid, and convenient technology could be used for ultrasensitive and robust visual mi RNA detection.     

Magnetic nanobead-based immunoassay for the simultaneous detection of aflatoxin B1 and ochratoxin A using upconversion nanoparticles as multicolor labels

A novel and sensitive immunoassay for the simultaneous detection of aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) in food samples was developed by using artificial antigen-modified magnetic nanoparticles (MNPs) as immunosensing probes and antibody functionalized upconversion nanoparticles (UCNPs) as signal probes. NaY_0.78F₄:Yb_0.2, Tm_0.02 and NaY_0.28F₄:Yb_0.7,Er_0.02 UCNPs were prepared and functionalized, respectively, with immobilized monoclonal anti-AFB₁ antibodies and anti-OTA antibodies as signal probes. Based on a competitive immunoassay format, the detection limit for both AFB₁ and OTA under optimal conditions was as low as 0.01 ng mL⁻¹, and the effective detection range was from 0.01 to 10 ng mL⁻¹. The proposed method was successfully applied to measure AFB₁ and OTA in naturally contaminated maize samples and compared to a commercially available ELISA method. The high sensitivity and selectivity of this method is due to the magnetic separation and concentration effect of the MNPs, the high sensitivity of the UCNPs, and the different emission lines of Yb/Tm and Yb/Er doped NaYF₄ UCNPs excited by 980 nm laser. Multicolor UCNPs have the potential to be used in other applications for detecting toxins in the field of food safety and other fields.     

Nanodiagnostic Method for Colorimetric Detection of Mycobacterium tuberculosis 16S rRNA

A nanodiagnostic method using nucleic acid sequence-based amplification (NASBA) and gold nanoparticle probes (AuNP probes) was developed for colorimetric detection of Mycobacterium tuberculosis. The primers targeting 16S rRNA were used for the amplification of mycobacterial RNA by the isothermal NASBA process. The amplicons were hybridized with specific gold nanoparticle probes. The RNA–DNA hybrids were colorimetrically detected by the accumulation of gold nanoparticles. Using this method, 10 CFU ml^?1 of M. tuberculosis was detected within less than 1 h. Results obtained from the clinical specimens showed 94.7% and 96% sensitivity and specificity, respectively. No interference was encountered in the amplification and detection of M. tuberculosis in the presence of non-target bacteria, confirming the specificity of the method.     

Comparison of three nonradioactive and a radioactive DNA probe for the detection of target DNA by DNA hybridization

The specificity and sensitivity of three methods for the preparation and detection of nonradioactive probe DNA (biotin-nick translation, biotin-photolabel, and antigen-chemical linkage) were evaluated and compared with a nick-translated³²P-labeled DNA probe in DNA hybridization studies. The DNA probes were prepared from a restriction fragment (HindIII-3) from bacteriophage P1 DNA, and target DNA consisted of purified phage P1 DNA or P1 prophage DNA in lysogens ofEscherichia coli. A probe concentration of 50 ng/ml resulted in clear detection with the three nonradioactiveHindIII-3 DNA probes, whereas the specificity of the³²P-HindIII-3 DNA probe was satisfactory at a concentration of 25 ng/ml. However, the detection of false positives was greater with the³²P-labeled probe. The sensitivity of the radiolabeled DNA probe was marginally greater than that of the nonradioactive probes in dot blot hybridizations with purified phage P1 DNA. However, when the preparation time, ease of use, safety, duration of storage, and expense were compared for the four methods of labeling, the nonradiolabeled probes were generally superior to the radiolabeled probe.     

Identification of single-point mutations in mycobacterial 16S rRNA sequences by confocal single-molecule fluorescence spectroscopy

We demonstrate the specific identification of single nucleotide polymorphism (SNP) responsible for rifampicin resistance of Mycobacterium tuberculosis applying fluorescently labeled DNA-hairpin structures (smart probes) in combination with single-molecule fluorescence spectroscopy. Smart probes are singly labeled hairpin-shaped oligonucleotides bearing a fluorescent dye at the 5′ end that is quenched by guanosine residues in the complementary stem. Upon hybridization to target sequences, a conformational change occurs, reflected in a strong increase in fluorescence intensity. An excess of unlabeled (‘cold’) oligonucleotides was used to prevent the formation of secondary structures in the target sequence and thus facilitates hybridization of smart probes. Applying standard ensemble fluorescence spectroscopy we demonstrate the identification of SNPs in PCR amplicons of mycobacterial rpoB gene fragments with a detection sensitivity of 10⁻⁸ M. To increase the detection sensitivity, confocal fluorescence microscopy was used to observe fluorescence bursts of individual smart probes freely diffusing through the detection volume. By measuring burst size, burst duration and fluorescence lifetime for each fluorescence burst the discrimination accuracy between closed and open (hybridized) smart probes could be substantially increased. The developed technique enables the identification of SNPs in 10⁻¹¹ M solutions of PCR amplicons from M.tuberculosis in only 100 s.     

Molecular Detection of <Emphasis Type="Italic">Penicillium griseofulvum</Emphasis> as the Coastal Pollution Indicator

Molecular methods were carried out to detect Penicillium griseofulvum, a dominant species related to heavy metal pollution, which was screened from marine contaminated sediments. Based on differences in internal transcribed spacer (ITS) sequences of Penicillium genus and specific isoamyl alcohol oxidase (IAO) sequences, species-specific primers AS1/RS4 and IAO1/IAO2 of Penicillium griseofulvum were designed and synthesized which were then employed in optimized PCR systems. The detection sensitivities were compared through ordinary PCR and nested-PCR using two pairs of primers, respectively. Both primer pairs could exclusively amplify destined DNA fragment from contaminated environmental samples in our researches. As for primers AS1/RS4, the detection sensitivity for spores (pure spore DNA) could be 10 fg/μl and 10 spores, respectively, and the detection sensitivity for the sediments was 10² spores/0.25 g sediments. While the detection sensitivity of IAO1/IAO2 primers was lower than that of AS1/RS4. Despite the difference in detection sensitivity, it is feasible that the species-specific primers could be used as probes for the detection of environmental pollution dominant species, Penicillium griseofulvum, since the frequency of occurrence and amount of this strain could preferably indicate the pollution degree.     

Peptides panned from a phage-displayed random peptide library are useful for the detection of Bacillus anthracis surrogates B. cereus 4342 and B. anthracis Sterne

Recent use of Bacillus anthracis as a bioweapon has highlighted the need for a sensitive monitoring system. Current bacterial detection tests use antibodies as bio-molecular recognition elements which have limitations with regard to time, specificity and sensitivity, creating the need for new and improved cost-effective high-affinity detection probes. In this study, we screened a commercially available bacteriophage-displayed random peptide library using Bacillus cereus 4342 cells as bait to identify peptides that could be used for detection of Bacillus. The method enabled us to identify two 12-amino acid consensus peptide sequences that specifically bind to B. cereus 4342 and B. anthracis Sterne, the nonpathogenic surrogates of B. anthracis strain. The two Bacillus-binding peptides (named BBP-1 and BBP-2) were synthesized with biotin tag to confirm their binding by four independent detection assays. Dot-blot analysis revealed that the peptides bind specifically to B. cereus 4342 and B. anthracis Sterne. Quantitative analysis of this interaction by ELISA and fluorometry demonstrated a detection sensitivity of 10² colony forming U/ml (CFU/ml) by both assays. When the peptides were used in combination with Qdots, the sensitivity was enhanced further by enabling detection of even a single bacterium by fluorescence microscopy. Immunoblot analysis and protein sequencing showed that BBP-1 and BBP-2 bound to the S-layer protein of B. anthracis Sterne. Overall, our findings validate the usefulness of synthetic versions of phage-derived peptides in combination with Qdot-liquid nanocrystals as high sensitivity bioprobes for various microbial detection platforms.     

32P- and biotin-labelled in vitro transcribed cRNA probes for the detection of potato spindle tuber viroid and chrysanthemum stunt viroid

Replacing nick-translated DNA probes by in vitro transcribed complementary RNA (cRNA) probes considerably increased the sensitivity of dot-blot detection tests of potato spindle tuber viroid and chrysanthemum stunt viroid. As compared to the limit of detection of 5–10 pg of viroid obtained with ³²P-labelled DNA probes, cRNA probes allow the detection of less than 1 pg of pure viroid. When labelled with biotin by incorporation of biotin-labelled ribonucleotides, the cRNA probes have a limit of detection of approximately 5 pg of purified viroid.     

Boronate probes for the detection of hydrogen peroxide release from human spermatozoa

Human spermatozoa are compromised by production of reactive oxygen species (ROS), and detection of ROS in spermatozoa is important for the diagnosis of male infertility. The probes 2′,7′-dichlorohydrofluorescein diacetate (DCFH), dihydroethidium (DHE), and MitoSOX red (MSR) are commonly used for detecting ROS by flow cytometry; however, these probes lack sensitivity to hydrogen peroxide (H₂O₂), which is particularly damaging to mammalian sperm cells. This study reports the synthesis and use of three aryl boronate probes, peroxyfluor-1 (PF1), carboxyperoxyfluor-1, and a novel probe, 2-(2-ethoxyethoxy)ethoxyperoxyfluor-1 (EEPF1), in human spermatozoa. PF1 and EEPF1 were effective at detecting H₂O₂ and peroxynitrite (ONOO⁻) produced by spermatozoa when stimulated with menadione or 4-hydroxynonenal. EEPF1 was more effective at detection of ROS in spermatozoa than DCFH, DHE, or MSR; furthermore it distinguished poorly motile sperm as shown by greater ROS production. EEPF1 should therefore have a significant role in the diagnosis of oxidative stress in male infertility, cryopreservation, age, lifestyle, and exposure to environmental toxicants.     

OX40-OX40L Interaction Promotes Proliferation and Activation of Lymphocytes via NFATc1 in ApoE-Deficient Mice

Background

Our previous studies have shown that OX40-OX40L interaction regulates the expression of nuclear factor of activated T cells c1(NFATc1) in ApoE^−/− mice during atherogenesis. The aim of this study was to investigate whether OX40-OX40L interaction promotes Th cell activation via NFATc1 in ApoE^−/− mice.

Methods and Results

The lymphocytes isolated from spleen of ApoE^−/− mice were cultured with anti-CD3 mAb in the presence or absence of anti-OX40 or anti-OX40L antibodies. The expression of NFATc1 mRNA and protein in isolated lymphocytes were measured by real time PCR (RT-PCR) and flow cytometry (FCM), respectively. The proliferation of lymphocytes was analyzed by MTT method,and the expression of IL-2, IL-4 and IFN-γ in the cultured cells and supernatant were measured by RT-PCR and enzyme-linked immunosorbent assary (ELISA), respectively. After stimulating OX40-OX40L signal pathway, the expression of NFATc1 and the proliferation of leukocytes were significantly increased. Anti-OX40L suppressed the expression of NFATc1 in lymphocytes of ApoE−/− mice. Anti-OX40L or the NFATc1 inhibitor (CsA) markedly suppressed the cell proliferation induced by anti-OX40. Moreover, the expression of IL-2 and IFN-γ was increased in lymphocytes induced by OX40-OX40L interaction. Blocking OX40-OX40L interaction or NFATc1 down-regulated the expression of IL-2 and IFN-γ, but didn’t alter the expression of IL-4 in supernatants.

Conclusion

These results suggest that OX40-OX40L interaction promotes the proliferation and activation of lymphocytes through NFATc1.     

Osthole inhibits osteoclasts formation and bone resorption by regulating NF-κB signaling and NFATc1 activations stimulated by RANKL

Chinese herbal medicine Fructus Cnidii has an outstanding effect on chronic lumbar pain and impotence, also has been used against osteoporosis with high frequency. Yet, the mechanisms of osthole, a derivative of Fructus Cnidii, on osteoclasts remains barely known. In this study, it was found out that osthole (10⁻⁶mol/L, 10⁻⁵mol/L) had the influence of inhibiting osteoclast formation and bone resorptive activities induced by receptor activator of nuclear factor κB ligand (RANKL), rather than affecting the viability of osteoclast-like cells. Furthermore, osthole could also inhibit the messenger RNA expressions of c-Src, tartrate-resistant acid phosphatase, β3-Integrin, matrix metallopeptidase 9, and cathepsin K. The results of the mechanistic study indicated that osthole regulated the nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) and nuclear factor-κB (NF-κB) activations following the RANKL stimulation. These findings suggested that the inhibitory effects of osthole were associated with restraining the activations of NFATc1 and NF-κB induced by RANKL. Thus osthole can be used as a potential treatment for abnormal bone-resorption related diseases.     

Detection of cyclin D1 mRNA by hybridization sensitive NIC–oligonucleotide probe

A large group of fluorescent hybridization probes, includes intercalating dyes for example thiazole orange (TO). Usually TO is coupled to nucleic acids post-synthetically which severely limits its use. Here, we have developed a phosphoramidite monomer, 10, and prepared a 2′-OMe-RNA probe, labeled with 5-(trans-N-hexen-1-yl-)-TO-2′-deoxy-uridine nucleoside, dU^TO, (Nucleoside bearing an Inter-Calating moiety, NIC), for selective mRNA detection. We investigated a series of 15-mer 2′-OMe-RNA probes, targeting the cyclin D1 mRNA, containing one or several dU^TO at various positions. dU^TO-2′-OMe-RNA exhibited up to 7-fold enhancement of TO emission intensity upon hybridization with the complementary RNA versus that of the oligomer alone. This NIC-probe was applied for the specific detection of a very small amount of a breast cancer marker, cyclin D1 mRNA, in total RNA extract from cancerous cells (250 ng/μl). Furthermore, this NIC-probe was found to be superior to our related NIF (Nucleoside with Intrinsic Fluorescence)-probe which could detect cyclin D1 mRNA target only at high concentrations (1840 ng/μl). Additionally, dU^T can be used as a monomer in solid-phase oligonucleotide synthesis, thus avoiding the need for post-synthetic modification of oligonucleotide probes. Hence, we propose dU^TO oligonucleotides, as hybridization probes for the detection of specific RNA in homogeneous solutions and for the diagnosis of breast cancer.