Fluorescence-based DHPLC for allelic quantification by single-nucleotide primer extension

We have investigated the possibility of determining quantitatively the alleles of binary DNA polymorphisms by single-nucleotide primer extension (SNuPE) and fluorescence-based DHPLC. Using a polymorphism of interest to our group, ROX-labeled dideoxy CTP (ROX-ddCTP) was incorporated at the 3' end of the primer annealed to the template adjacent to the polymorphic site. The primer extension product was then resolved from the unincorporated dye terminator by ion-pair reversed-phase liquid chromatography. The signal intensity of incorporated ROX-ddCTP correlated well over one order of magnitude with the relative amount of the C-allele present in the genomic DNA template. We conclude that SNuPE, when combined with fluorescence-based DHPLC, can accurately determine the relative molar proportion of one allele in total DNA.     

Quantitative methylation analysis using methylation-sensitive single-nucleotide primer extension (Ms-SNuPE)

Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) is a technique that allows for rapid and simultaneous quantitation of the degree of methylation at several CpG sites. Treatment of genomic DNA with sodium bisulfite is used to convert unmethylated cytosine to uracil while leaving 5-methylcytosine unchanged. A strand-specific polymerase chain reaction product is then generated to provide a suitable DNA template for quantitative methylation analysis using Ms-SNuPE. Single-nucleotide primer extension is performed with oligonucleotide(s) designed to hybridize immediately upstream of the CpG site(s) being analyzed. The Ms-SNuPE technique can be adapted for high-throughput methylation analysis and therefore represents a novel approach for rapid quantitation of cytosine methylation suitable for a wide range of biological investigations.     

Simultaneous detection of dual proteins using quantum dots coated silica nanoparticles as labels

Simultaneous detection of multianalytes associated with a particular cancer is beneficial for disease diagnosis. Here, a facile immunosensing strategy was designed to allow simultaneous electrochemical detection of dual proteins, in a single run. CdSe and PbS water-soluble quantum dots (QDs) were prepared and coated on monodisperse silica nanoparticles as labels for proteins detection. Rabbit immunoglobulin G antigen (IgG) and carcinoembryonic antigen (CEA) were chosen as model proteins for analysis. After a typical sandwich immunoassay, CdSe and PbS QDs labels were introduced onto the Au substrates' surface, which were then dissolved and could be simultaneously monitored by square-wave-voltammetric (SWV) stripping measurements. Under selected conditions, IgG and CEA could be assayed in the ranges of 0.05-40 ng mL(-1) and 0.05-25 ng mL(-1), respectively. The proposed method possessed high sensitivity, good precision, and satisfactory reproducibility and regeneration.     

量子点荧光标记技术的研究热点及面临的挑战

半导体量子点作为新型荧光标记物,在生物医学领域具有重要应用．与传统的有机染料及荧光蛋白等荧光标记物相比,半导体量子点具有发光颜色可调、激发范围宽、发射光谱窄、化学及光稳定性好、表面化学丰富以及生物偶联技术成熟等诸多优势,为生命体系的靶向示踪,高灵敏、原位、实时、动态荧光成像,DNA及蛋白质检测,靶向药物,临床医学,生物芯片和传感器等研究提供了新的发展契机．基于作者在半导体量子点生物荧光成像和安全性评价研究的基础,综述了半导体量子点荧光标记物在生命科学与医学领域应用的研究热点,并对半导体量子点荧光标记技术走向实用面临的挑战进行了评述．     

Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) for quantitative measurement of DNA methylation

Methylation-sensitive single-nucleotide primer extension (Ms-SNuPE) is a technique that can be used for rapid quantitation of methylation at individual CpG sites. Treatment of genomic DNA with sodium bisulfite is used to convert unmethylated Cytosine to Uracil while leaving 5-methylcytosine unaltered. Strand-specific PCR is performed to generate a DNA template for quantitative methylation analysis using Ms-SNuPE. SNuPE is then performed with oligonucleotide(s) designed to hybridize immediately upstream of the CpG site(s) being interrogated. Reaction products are electrophoresed on polyacrylamide gels for visualization and quantitation by phosphorimage analysis. The Ms-SNuPE technique is similar to other quantitative assays that use bisulfite treatment of genomic DNA to discriminate unmethylated from methylated Cytosines (i.e., COBRA, pyrosequencing). Ms-SNuPE can be used for high-throughput methylation analysis and rapid quantitation of Cytosine methylation suitable for a wide range of biological investigations, such as checking aberrant methylation changes during tumorigenesis, monitoring methylation changes induced by DNA methylation inhibitors or for measuring hemimethylation. Approximately two to four CpG sites can be interrogated in up to 40 samples by Ms-SNuPE in less than 5 h, after PCR amplification of the desired target sequence and preparation of PCR amplicons.     

Multienzyme-nanoparticles amplification for sensitive virus genotyping in microfluidic microbeads array using Au nanoparticle probes and quantum dots as labels

A novel microfluidic device with microbeads array was developed and sensitive genotyping of human papillomavirus was demonstrated using a multiple-enzyme labeled oligonucleotide-Au nanoparticle bioconjugate as the detection tool. This method utilizes microbeads as sensing platform that was functionalized with the capture probes and modified electron rich proteins, and uses the horseradish peroxidase (HRP)-functionalized gold nanoparticles as label with a secondary DNA probe. The functionalized microbeads were independently introduced into the arrayed chambers using the loading chip slab. A single channel was used to generate weir structures to confine the microbeads and make the beads array accessible by microfluidics. Through "sandwich" hybridization, the enzyme-functionalized Au nanoparticles labels were brought close to the surface of microbeads. The oxidation of biotin-tyramine by hydrogen peroxide resulted in the deposition of multiple biotin moieties onto the surface of beads. This deposition is markedly increased in the presence of immobilized electron rich proteins. Streptavidin-labeled quantum dots were then allowed to bind to the deposited biotin moieties and displayed the signal. Enhanced detection sensitivity was achieved where the large surface area of Au nanoparticle carriers increased the amount HRP bound per sandwiched hybridization. The on-chip genotyping method could discriminate as low as 1fmol/L (10zmol/chip, SNR>3) synthesized HPV oligonucleotides DNA. The chip-based signal enhancement of the amplified assay resulted in 1000 times higher sensitivity than that of off-chip test. In addition, this on-chip format could discriminate and genotype 10copies/μL HPV genomic DNA using the PCR products. These results demonstrated that this on-chip approach can achieve highly sensitive detection and genotyping of target DNA and can be further developed for detection of disease-related biomolecules at the lowest level at their earliest incidence.     

Differential replication timing of X-linked genes measured by a novel method using single-nucleotide primer extension.

The ratio of two differentially replicating alleles is not constant during S phase. Using this fact, we have developed a method for determining allele-specific replication timing for alleles differing by at least a single base pair. Unsynchronized cells in tissue culture are first sorted into fractions based on DNA content as a measure of position in S phase. DNA is purified from each fraction and used for PCR with primers that bracket the allelic difference, amplifying both alleles. The ratio of alleles in the amplified product is then determined by a single nucleotide primer extension (SNuPE) assay, modified as described [Singer-Sam,J. and Riggs,A.D. (1993) Methods Enzymol., 225, 344-351]. We report here use of this SNuPE-based method to analyze replication timing of two X-linked genes, Pgk-1 and Xist, as well as the autosomal gene Gabra-6. We have found that the two alleles of the Gabra-6 gene replicate synchronously, as expected; similarly, the active allele of the Pgk-1 gene on the active X chromosome (Xa) replicates early relative to the silent allele on the inactive X chromosome (Xi). In contrast, the expressed allele of the Xist gene, which is on the Xi, replicates late relative to the silent allele on the Xa.     

A robust and fast bacteria counting method using CdSe/ZnS core/shell quantum dots as labels

A rapid, sensitive fluorescence measurement method for detecting the bacterial count using CdSe/ZnS as a fluorescence marker was described. High-quality CdSe/ZnS nanocrystals were synthesized and successfully conjugated with bacteria. The fluorescence intensity was proportional to bacterial count in the range of 10²–10⁸ CFU/mL and the low detection limit was 10² CFU/mL.     

Quantum dots versus organic dyes as fluorescent labels

Suitable labels are at the core of Luminescence and fluorescence imaging and sensing. One of the most exciting, yet also controversial, advances in label technology is the emerging development of quantum dots (QDs)--inorganic nanocrystals with unique optical and chemical properties but complicated surface chemistry--as in vitro and in vivo fluorophores. Here we compare and evaluate the differences in physicochemical properties of common fluorescent labels, focusing on traditional organic dyes and QDs. Our aim is to provide a better understanding of the advantages and limitations of both classes of chromophores, to facilitate label choice and to address future challenges in the rational design and manipulation of QD labels.     

Quantum dots as strain- and metabolism-specific microbiological labels

Biologically conjugated quantum dots (QDs) have shown great promise as multiwavelength fluorescent labels for on-chip bioassays and eukaryotic cells. However, use of these photoluminescent nanocrystals in bacteria has not previously been reported, and their large size (3 to 10 nm) makes it unclear whether they inhibit bacterial recognition of attached molecules and whether they are able to pass through bacterial cell walls. Here we describe the use of conjugated CdSe QDs for strain- and metabolism-specific microbial labeling in a wide variety of bacteria and fungi, and our analysis was geared toward using receptors for a conjugated biomolecule that are present and active on the organism's surface. While cell surface molecules, such as glycoproteins, make excellent targets for conjugated QDs, internal labeling is inconsistent and leads to large spectral shifts compared with the original fluorescence, suggesting that there is breakup or dissolution of the QDs. Transmission electron microscopy of whole mounts and thin sections confirmed that bacteria are able to extract Cd and Se from QDs in a fashion dependent upon the QD surface conjugate.     

Exploring permeability of Escherichia coli competence using quantum dots as fluorescent probes

Though people had recognized the pivotal function of CaCl(2) during DNA transformation into Escherichia coli, the mechanism of divalent Ca(2+) cation inducing E. coli competence development is still unknowable. Quantum dots (QDs), as a new fluorescent probe, being applied in biology research, had aroused great interest. We explored the penetrability of E. coli competent cells membrane using QDs and proved directly that competent cells were more permeable than that of noncompetent. The results are significant on understanding the problems of the microbiological genetics.     

Targeted nuclear delivery using peptide-coated quantum dots

Core/shell quantum dots (CdSe/Zns) conjugated with various nuclear localization signaling (NLS) peptides, which could facilitate the transportation of quantum dots across the plasma membrane into the nucleus, have been utilized to investigate the uptake mechanism of targeted delivery. Because of their brightness and photostability, it was possible to trace the trajectories of individual quantum dots in living cells using both confocal and total internal reflection microscopes. We found that, when the quantum dots were added to a cell culture, the peptide-coated quantum dots entered the cell nucleus while the uncoated quantum dots remained in the cytoplasm. At 8 nM, most of the peptide coated quantum dots were found in the cytoplasm due to aggregation. However, at a lower concentration (0.08 nM), approximately 25% of the NLS peptide-coated quantum dots entered the cell nucleus. We also found that some quantum dots without NLS coating could also enter the nucleus, suggesting that the size of the quantum dots may play an important role in such a process.     

Magnetic bead-based DNA detection with multi-layers quantum dots labeling for rapid detection of Escherichia coli O157:H7

This work demonstrated the feasibility of detecting 250zM Escherichia coli O157:H7 eaeA target DNA by using a magnetic bead-based DNA detection assay with designed labeling strategy within 40-60min. The magnetic beads were used as the solid support for the binding probe and isolated the target DNA from the sample. The detection signals could be amplified from the multi-layers biotin-streptavidin conjugated quantum dots based on binding with specific designed biotinlyted linker. This assay method would provide a simple, rapid, and ultra-sensitive detection method for DNA or other biomolecular analysis.     

Microfluidic separation of (S)-ibuprofen using enzymatic reaction

This study was investigated for the enantioselective separation of (S)-ibuprofen using the ionic liquid in the microfluidic device. A stable and thin ionic liquid flow (ILF) was made by controlling the flow rate of the ILF in the microfluidic channel. In addition, coupling lipase as a biocatalyst with the ILF based on the microfluidic device showed the facilitative and selective transport of (S)-ibuprofen across the ILF, indicating successful optical resolution of a racemic mixture. Subsequently, the enantioselectivity was evaluated in the transport ratio (η) of (R)- and (S)-ibuprofen, the optical resolution ratio (α) and enantiomeric excess of (S)-ibuprofen (ee_S).     

Novel fluoroimmunoassays for detecting ochratoxin A using CdTe quantum dots

Novel direct and indirect competitive fluorescence‐linked immunosorbent assays (c FLISA and ic FLISA) for detection of ochratoxin A (OTA) were described using CdTe quantum dots (QDs) as fluorescent label. CdTe QDs were successfully synthesized, which had an emission wavelength of 615 nm. The high purity monoclonal antibody against OTA was prepared through cell thawing and the octylic acid‐ammonium sulfate method. The OTA MAbs were successfully coupled with CdTe QDs, and which also retained the original biological activity. The 50% inhibition values (IC₅₀) of the c FLISA and ic FLISA were 0.630 ng/mL, 0.234 ng/mL, the limits of detection (LOD) were 7.06 × 10^–3 and 4.15 × 10^–3 ng/mL, and detection ranges were 7.06 × 10^–3 ? 18.34 ng/mL and 4.15 × 10^–3 ? 4.88 ng/mL, in‐order. The recoveries were 96.0–104.7% along with coefficients of variation (CVs) below 10%. The FLISA provided novel method for determination of OTA and the potential of MAb–CdTe QDs for the establishment of fluorescent immunochromatographic test strip.

     

Semiconductor quantum dots as contrast agents for whole animal imaging

Recent developments in quantum dot (QD) technology have paved the way for using QDs as optical contrast agents for in vivo imaging. Pioneering papers showed the use of QDs as luminescent contrast agents for imaging cancer and guiding cancer surgery. The possible future use of QDs for clinical applications is expected to have a significant impact, however many challenges in this field have yet to be overcome.     

A simple electrochemical aptasensor for ultrasensitive protein detection using cyclic target-induced primer extension

A simple electrochemical aptasensor was developed for ultrasensitive protein detection by combining a novel strategy of cyclic target-induced primer extension (CTIPE) with an aptamer-hairpin probe and enzyme-amplified electrochemical readout. In the presence of protein target, the immobilized aptamer-hairpin probe recognized the protein to trigger primer extension reaction by target-induced conformational transition, which released the protein from replicated DNA duplex. The released target could cyclically bind with other aptamer-hairpin probes and trigger new primer extension, leading to formation of numerous biotin-tagged DNA duplex, which significantly amplified the protein recognition event and facilitated the subsequent enzymatic signal enhancement, leading to an ultrasensitive electrochemical aptasensor. Using human vascular endothelial growth factor as a model protein, the designed aptasensor could detect protein down to 0.82 pg mL(-1) with a linear range from 1 pg mL(-1) to 1 ng mL(-1). The proposed aptasensor was amenable to quantification of protein in complex biological matrixes, and would become a simple and powerful tool for bioanalysis and clinic diagnostic application.     

Real-time nanoscopy by using blinking enhanced quantum dots

Superresolution optical microscopy (nanoscopy) is of current interest in many biological fields. Superresolution optical fluctuation imaging, which utilizes higher-order cumulant of fluorescence temporal fluctuations, is an excellent method for nanoscopy, as it requires neither complicated optics nor illuminations. However, it does need an impractical number of images for real-time observation. Here, we achieved real-time nanoscopy by modifying superresolution optical fluctuation imaging and enhancing the fluctuation of quantum dots. Our developed quantum dots have higher blinking than commercially available ones. The fluctuation of the blinking improved the resolution when using a variance calculation for each pixel instead of a cumulant calculation. This enabled us to obtain microscopic images with 90-nm and 80-ms spatial-temporal resolution by using a conventional fluorescence microscope without any optics or devices.