Qingge Li

Aptamer-Based Microfluidic Device for Enrichment, Sorting, and Detection of Multiple Cancer Cells

The ability to diagnose cancer based on the detection of rare cancer cells in blood or other bodily fluids is a significant challenge. To address this challenge, we have developed a microfluidic device that can simultaneously sort, enrich, and then detect multiple types of cancer cells from a complex sample. The device, which is made from poly(dimethylsiloxane) (PDMS), implements cell-affinity chromatography based on the selective cell-capture of immobilized DNA-aptamers and yields a 135-fold enrichment of rare cells in a single run. This enrichment is achieved because the height of the channel is on the order of a cell diameter. The sorted cells grow at the comparable rate as cultured cells and are 96% pure based on flow cytometry determination. Thus, by using our aptamer based device, cell capture is achieved simply and inexpensively, with no sample pretreatment before cell analysis. Enrichment and detection of multiple rare cancer cells can be used to detect cancers at the early stages, diagnose metastatic relapse, stratify patients for therapeutic purposes, monitor response to drugs and therapies, track tumor progression, and gain a deeper understanding of the biology of circulating tumor cells (CTCs).

Quantitative Analysis of the Role Played by Poly(vinylpyrrolidone) in Seed-Mediated Growth of Ag Nanocrystals

This article presents a quantitative analysis of the role played by poly(vinylpyrrolidone) (PVP) in seed-mediated growth of Ag nanocrystals. Starting from Ag nanocubes encased by {100} facets as the seeds, the resultant nanocrystals could take different shapes depending on the concentration of PVP in the solution. If the concentration was above a critical value, the seeds simply grew into larger cubes still enclosed by {100} facets. When the concentration fell below a critical value, the seeds would evolve into cuboctahedrons enclosed by a mix of {100} and {111} facets and eventually octahedrons completely covered by {111} facets. We derived the coverage density of PVP on Ag(100) surface by combining the results from two measurements: (i) cubic seeds were followed to grow at a fixed initial concentration of PVP to find out when {111} facets started to appear on the surface, and (ii) cubic seeds were allowed to grow at reduced initial concentrations of PVP to see at which concentration {111} facets started to appear from the very beginning. We could calculate the coverage density of PVP from the differences in PVP concentration and the total surface area of Ag nanocubes between these two samples. The coverage density was found to be 140 and 30 repeating units per nm(2) for PVP of 55,000 and 10,000 g/mol in molecular weight, respectively, for cubic seeds of 40 nm in edge length. These values dropped slightly to 100 and 20 repeating units per nm(2), respectively, when 100 nm Ag cubes were used as the seeds.

Silver Nanocrystals with Concave Surfaces and Their Optical and Surface-Enhanced Raman Scattering Properties

Shaped and dimpled: Silver nanocrystals enclosed by concave surfaces and thus high-index facets have been prepared by simply controlling the growth habit of Ag cubic seeds. Four types of concave nanocrystals, including octahedron, cube, octapod, and trisoctahedron, were obtained (see picture).

Successive Deposition of Silver on Silver Nanoplates: Lateral versus Vertical Growth

NSF [DMR-0804088, ECS-0335765]; Washington University in St. Louis; Ministry of Education, Science and Technology [R32-20031]; China Scholarship Council

Lateral Flow Immunoassay Using Europium Chelate-Loaded Silica Nanoparticles as Labels

BACKGROUND Despite their ease of use, lateral flow immunoassays (LFIAs) often suffer from poor quantitative discrimination and low analytical sensitivity. We explored the use of a novel class of europium chelate-loaded silica nanoparticles as labels to overcome these limitations. METHODS Antibodies were covalently conjugated onto europium chelate-loaded silica nanoparticles with dextran as a linker. The resulting conjugates were used as labels in LFIA for detection of hepatitis B surface antigen (HBsAg). We performed quantification with a digital camera and Adobe Photoshop software. We also used 286 clinical samples to compare the proposed method with a quantitative ELISA. RESULTS A detection limit of 0.03 microg/L was achieved, which was 100 times lower than the colloidal gold-based LFIAs and lower than ELISA. A precise quantitative dose-response curve was obtained, and the linear measurement range was 0.05-3.13 microg/L, within which the CVs were 2.3%-10.4%. Regression analysis of LFIA on ELISA results gave: log (LFIA) = -0.14 log (ELISA) + 1.03 microg/L with r = 0.99 for the quantification of HBsAg in 35 positive serum samples. Complete agreement was observed for the qualitative comparison of 286 clinical samples assayed with LFIA and ELISA. CONCLUSIONS Europium chelate-loaded silica nanoparticle labels have great potential to improve LFIAs, making them useful not only for simple screening applications but also for more sensitive and quantitative immunoassays.

Fluorescent Probe-Based Lateral Flow Assay for Multiplex Nucleic Acid Detection

Here we report a rapid, low cost, and disposable dipstick-type DNA biosensor that enables multiplex detection in a single assay. The fluorescent probes labeled with different fluorophores were introduced into the lateral flow nucleic acid testing system. In combination with multiple immobilized probes arranged in an array formant on the membrane, a dual-color fluorescent lateral flow DNA biosensor was developed using a portable fluorescence reader. Up to 13 human papillomavirus types could be detected simultaneously by a single-step operation in less than 30 min after linear-after-the-exponential (LATE)-PCR. The sensitivity was determined to be 10-10(2) copies plasmid DNA/μL. The specificity study showed no cross-reactivity among the 31 different common HPV types. In the clinical validation, 95.3% overall agreement showed very good potential for this method in the clinical application when compared to a commercial kit.

Multiplex Fluorescence Melting Curve Analysis for Mutation Detection with Dual-Labeled, Self-Quenched Probes

Probe-based fluorescence melting curve analysis (FMCA) is a powerful tool for mutation detection based on melting temperature generated by thermal denaturation of the probe-target hybrid. Nevertheless, the color multiplexing, probe design, and cross-platform compatibility remain to be limited by using existing probe chemistries. We hereby explored two dual-labeled, self-quenched probes, TaqMan and shared-stem molecular beacons, in their ability to conduct FMCA. Both probes could be directly used for FMCA and readily integrated with closed-tube amplicon hybridization under asymmetric PCR conditions. Improved flexibility of FMCA by using these probes was illustrated in three representative applications of FMCA: mutation scanning, mutation identification and mutation genotyping, all of which achieved improved color-multiplexing with easy probe design and versatile probe combination and all were validated with a large number of real clinical samples. The universal cross-platform compatibility of these probes-based FMCA was also demonstrated by a 4-color mutation genotyping assay performed on five different real-time PCR instruments. The dual-labeled, self-quenched probes offered unprecedented combined advantage of enhanced multiplexing, improved flexibility in probe design, and expanded cross-platform compatibility, which would substantially improve FMCA in mutation detection of various applications.

An enzyme-sensitive probe for photoacoustic imaging and fluorescence detection of protease activity

A gold nanocage and dye conjugate has been demonstrated for use with photoacoustic imaging and fluorescence detection of protease activity. The detection sensitivity could be maximized by using gold nanocages with a localized surface plasmon resonance peak away from the emission peak of the dye. These hybrids can be potentially used as multimodal contrast agents for molecular imaging.

Tandem conjugation of enzyme and antibody on silica nanoparticle for enzyme immunoassay

We present a new type of enzyme-antibody conjugate that simplifies the labeling procedure and increases the sensitivity of enzyme-linked immunosorbent assay (ELISA). The conjugates were prepared through layer-by-layer immobilization of enzyme and antibody on a silica nanoparticle scaffold. A maximal amount of enzyme was immobilized on the nanoparticle, followed by antibody linkage through Dextran 500. The conjugate could be easily purified from unreacted reagents by simple centrifugations. In comparison with the conventional antibody-enzyme conjugate used in ELISA, which often has one or two enzyme molecules per antibody, the new type of conjugate contained more enzyme molecules per antibody and provided a much higher signal and increased sensitivity. When used in an ELISA detection of the hepatitis B surface antigen (HBsAg), the detection limit was three times lower than that of the commercially available ELISA kit.

Quadruplex Real-Time PCR Assay for Detection and Identification of Vibrio cholerae O1 and O139 Strains and Determination of Their Toxigenic Potential

ABSTRACT Vibrio cholerae is a natural inhabitant of the aquatic environment. However, its toxigenic strains can cause potentially life-threatening diarrhea. A quadruplex real-time PCR assay targeting four genes, the cholera toxin gene (ctxA), the hemolysin gene (hlyA), O1-specific rfb, and O139-specific rfb, was developed for detection and differentiation of O1, O139, and non-O1, non-O139 strains and for prediction of their toxigenic potential. The specificity of the assay was 100% when tested against 70 strains of V. cholerae and 31 strains of non-V. cholerae organisms. The analytical sensitivity for detection of toxigenic V. cholerae O1 and O139 was 2 CFU per reaction with cells from pure culture. When the assay was tested with inoculated water from bullfrog feeding ponds, 10 CFU/ml could reliably be detected after culture for 3 h. The assay was more sensitive than the immunochromatographic assay and culture method when tested against 89 bullfrog samples and 68 water samples from bullfrog feeding ponds. The applicability of this assay was confirmed in a case study involving 15 bullfrog samples, from which two mixtures of nontoxigenic O1 and toxigenic non-O1/non-O139 strains were detected and differentiated. These data indicate that the quadruplex real-time PCR assay can both rapidly and accurately detect/identify V. cholerae and reliably predict the toxigenic potential of strains detected.