Fast magnetic isolation of simple sequence repeat markers in microfluidic channels.

Simple sequence repeat (SSR) markers are widely used for genome mapping, genetic diversity characterization and medical diagnosis. The fast isolation by AFLP of sequence containing repeats (FIASCO) is a powerful method for SSR marker isolation, but it is laborious, costly, and time consuming and requires multiple rounds of washing. Here, we report a superparamagnetic bead (SPMB)-based FIASCO method in a magnetic field controllable microfluidic chip (MFCM-Chip). This method dramatically reduces the assay time by 4.25-fold and reduces the quantity of magnetic beads and probes by 10-fold through the magnetic capture of (AG)n-containing fragments from Herba Leonuri, followed by washing and eluting on a microchip. The feasibility of this method was further evaluated by PCR and sequencing, and the results showed that the proportion of fragments containing SSRs was 89%, confirming that this platform is a fast and efficient method for SSR marker isolation. This cost-effective platform will make the powerful FIASCO technique more accessible for routine use with a wide variety of materials.

Surface labeling of enveloped viruses assisted by host cells.

Labeling of virus opens new pathways for the understanding of viruses themselves and facilitates the utilization of viruses in modern biology, medicine, and materials. Based on the characteristic that viruses hijack their host cellular machineries to survive and reproduce themselves, a host-cell-assisted strategy is proposed to label enveloped viruses. By simply feeding Vero cells with commercial 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (sodium salt) (Biotin-Cap-PE), we obtained biotinylated Vero cells whose membrane systems were modified with biotin. Subsequently, pseudorabies viruses (PrV) were cultivated in the biotinylated Vero cells, and the PrV progenies were spontaneously labeled with Biotin-Cap-PE during viral natural assembly process. Since the viral natural assembly process was employed for the labeling, potential threats of genetic engineering and difficulties in keeping viral natural bioactivity were avoided. Importantly, this labeling strategy for enveloped virus greatly reduces the technical complexity and allows researchers from different backgrounds to apply it for their specified demands.

One-to-one quantum dot-labeled single long DNA probes.

Quantum dots (QDs) have been received most attention due to their unique properties. Constructing QDs conjugated with certain number of biomolecules is considered as one of the most important research goals in nanobiotechnology. In this study, we report polymerase chain reaction (PCR) amplification of primer oligonucleotides bound to QDs, termed as QD-based PCR. Characterization of QD-based PCR products by gel electrophoresis and atomic force microscopy showed that QD-labeled long DNA strands were synthesized and only a single long DNA strand was conjugated with a QD. The QD-based PCR products still kept fluorescence properties. Moreover, the one-to-one QD-labeled long DNA conjugates as probes could detect a single-copy gene on maize chromosomes by fluorescence in situ hybridization. Labeling a single QD to a single long DNA will make detection of small single-copy DNA fragments, quantitative detection and single molecule imaging come true by nanotechnology, and it will promote medical diagnosis and basic biological research as well as nano-material fabrication.

PEG-interspersed nitrilotriacetic acid-functionalized quantum dots for site-specific labeling of prion proteins expressed on cell surfaces.

A strategy has been put forward to fabricate PEG-interspersed nitrilotriacetic acid (NTA)-functionalized QDs by one-step self-assembly using a mixture of self-synthesized NTA-terminated amphiphilic polymer and 1,2-Distearoyl-sn-Glycero-3-Phosphoethanolamine-N-[Carboxy(Polyethylene Glycol)2000] (DSPE-PEG-COOH). The process was highly reproducible for facile functionalization of QDs via simultaneous self-assembly of biocompatible PEG molecules onto their surface. An optimized molar ratio of NTA-terminated amphiphilic polymer to DSPE-PEG-COOH was used to obtain NTA-functionalized QDs for site-specific labeling of prion proteins (PrP(C)) expressed on cell surfaces. Fabricated NTA-functionalized QDs can be a good candidate used for real-time visualization of PrP(C) in single live cells so as to clarify the nosogenesis of pathogenic scrapie prion protein (PrP(Sc)).

Direct fluorescence in situ hybridization (FISH) in Escherichia coli with a target-specific quantum dot-based molecular beacon.

Quantum dots (QDs) are inorganic fluorescent nanocrystals with excellent properties such as tunable emission spectra and photo-bleaching resistance compared with organic dyes, which make them appropriate for applications in molecular beacons. In this work, quantum dot-based molecular beacons (QD-based MBs) were fabricated to specifically detect ß-lactamase genes located in pUC18 which were responsible for antibiotic resistance in bacteria Escherichia coli (E. coli) DH5α. QD-based MBs were constructed by conjugating mercaptoacetic acid-quantum dots (MAA-QDs) with black hole quencher 2 (BHQ2) labeled thiol DNA vial metal-thiol bonds. Two types of molecular beacons, double-strands beacons and hairpin beacons, were observed in product characterization by gel electrophoresis. Using QD-based MBs, one-step FISH in tiny bacteria DH5α was realized for the first time. QD-based MBs retained their bioactivity when hybridizing with complementary target DNA, which showed excellent advantages of eliminating background noise caused by adsorption of non-specific bioprobes and achieving clearer focus of genes in plasmids pUC18, and capability of bacterial cell penetration and signal specificity in one-step in situ hybridization.

Clickable gold nanoparticles as the building block of nanobioprobes.

A new method of fabricating "clickable" gold nanoparticles that could be used as the building block of nanobioprobes was described. On the basis of a well-developed strategy of encapsulating hydrophobic nanoparticles with a layer of amphiphilic polymers, cheap, easily prepared graft polymer was used as a modifier to prepare monodisperse azide-functionalized gold nanoparticles (AuNPs), which showed good stability in physiological solution. By conjugation with alkyne functional horseradish peroxidase (HRP) via click chemistry under mild conditions, the azide-AuNPs have demonstrated their potential in the fabrication of stable, bioactive nanobioprobes. Some critical problems in the fabrication of nanobioprobes, such as how to detect the number of bound biomolecules on nanoparticles and evaluate the bioactivities of nanobioprobes, are discussed in detail.

Color-tunable fluorescent-magnetic core/shell multifunctional nanocrystals.

We have developed a convenient strategy for preparing color-tunable fluorescent-magnetic core/shell multifunctional nanocrystals, which exhibit excellent photoluminescence (PL) properties (fluorescing tunably from 550 nm to 630 nm by modifying the shell thickness) and ferromagnetic material properties (a magnetization of 4.4 emu g(-1) and a coercivity of 95 Oe).

Facile preparation of low cytotoxicity fluorescent carbon nanocrystals by electrooxidation of graphite.

A simple and facile method was developed to prepare fluorescent carbon nanocrystals (CNCs) with low cytotoxicity and no photobleaching, by electrooxidation of graphite in aqueous solution.