Jiao Hu

Quick-Response Magnetic Nanospheres for Rapid, Efficient Capture and Sensitive Detection of Circulating Tumor Cells

The study on circulating tumor cells (CTCs) has great significance for cancer prognosis, treatment monitoring, and metastasis diagnosis, in which isolation and enrichment of CTCs are key steps due to their extremely low concentration in peripheral blood. Herein, magnetic nanospheres (MNs) were fabricated by a convenient and highly controllable layer-by-layer assembly method. The MNs were nanosized with fast magnetic response, and nearly all of the MNs could be captured by 1 min attraction with a commercial magnetic scaffold. In addition, the MNs were very stable without aggregation or precipitation in whole blood and could be re-collected nearly at 100% in a monodisperse state. Modified with anti-epithelial-cell-adhesion-molecule (EpCAM) antibody, the obtained immunomagnetic nanospheres (IMNs) successfully captured extremely rare tumor cells in whole blood with an efficiency of more than 94% via only a 5 min incubation. Moreover, the isolated cells remained viable at 90.5 ± 1.2%, and they could be directly used for culture, reverse transcription-polymerase chain reaction (RT-PCR), and immunocytochemistry (ICC) identification. ICC identification and enumeration of the tumor cells in the same blood samples showed high sensitivity and good reproducibility. Furthermore, the IMNs were successfully applied to the isolation and detection of CTCs in cancer patient peripheral blood samples, and even one CTC in the whole blood sample was able to be detected, which suggested they would be a promising tool for CTC enrichment and detection.

Optically encoded multifunctional nanospheres for one-pot separation and detection of multiplex DNA sequences.

In this study, we report a simple method for simultaneous detection of multiplex DNA sequences, including complementary DNA (cDNA) sequences of HIV and HCV, DNA sequence of HBV, with QDs-encoded fluorescent nanospheres and nano-γ-Fe2O3-coated magnetic nanospheres. Detection was achieved on a fluorescence spectrophotometer without additional auxiliary instruments, and the detection limit was about 100 pM. Here, QDs-encoded fluorescent nanospheres (FNS) with different photoluminescent properties, and magnetic nanospheres (MNS) were separately fabricated by stepwise assembly of hydrophobic QDs or nano-γ-Fe2O3 on the surface of branched poly(ethylene imine) (PEI)-coated nanospheres in precisely controlled amounts, finally followed by silica encapsulation. FNS-labeled probe DNAs and MNS-labeled capture DNAs were used to hybridize with the corresponding targets at the same time. After magnetic separation, the sandwich-structured adducts were measured by fluorescence spectrophotometry. The results indicated that the targets could be detected with high sensitivity. This method is convenient, fast enough, and capable of high anti-interference. Therefore, it is expected to be used for simultaneous detection and separation of multiple targets at high levels of purity and throughput.

Engineered decomposable multifunctional nanobioprobes for capture and release of rare cancer cells.

Early detection and isolation of circulating tumor cells (CTCs) can provide helpful information for diagnosis, and functional readouts of CTCs can give deep insight into tumor biology. In this work, we presented a new strategy for simple isolation and release of CTCs using engineered nanobioprobes. The nanobioprobes were constructed by Ca(2+)-assisted layer-by-layer assembly of alginate onto the surface of fluorescent-magnetic nanospheres, followed by immobilization of biotin-labeled anti-EpCAM. As-prepared anti-EpCAM-functionalized nanobioprobes were characterized with integrated features of anti-EpCAM-directed specific recognition, fluorescent magnetic-driven cell capture, and EDTA-assisted cell release, which can specifically recognize 10(2) SK-BR-3 cells spiked in 1 mL of lysed blood or human whole blood samples with 89% and 86% capture efficiency, respectively. Our proof-of-concept experiments demonstrated that 65% of captured SK-BR-3 cells were released after EDTA treatment, and nearly 70% of released SK-BR-3 cells kept their viability, which may facilitate molecular profiling and functional readouts of CTCs.

Sensitive and Quantitative Detection of C-Reaction Protein Based on Immunofluorescent Nanospheres Coupled with Lateral Flow Test Strip

Sensitive and quantitative detection of protein biomarkers with a point-of-care (POC) assay is significant for early diagnosis, treatment, and prognosis of diseases. In this paper, a quantitative lateral flow assay with high sensitivity for protein biomarkers was established by utilizing fluorescent nanospheres (FNs) as reporters. Each fluorescent nanosphere (FN) contains 332 ± 8 CdSe/ZnS quantum dots (QDs), leading to its superstrong luminescence, 380-fold higher than that of one QD. Then a detection limit of 27.8 pM C-reaction protein (CRP) could be achieved with an immunofluorescent nanosphere (IFN)-based lateral flow test strip. The assay was 257-fold more sensitive than that with a conventional Au-based lateral flow test strip for CRP detection. Besides, the fluorescence intensity of FNs and bioactivity of IFNs were stable during 6 months of storage. Hence, the assay owns good reproducibility (intra-assay variability of 5.3% and interassay variability of 6.6%). Furthermore, other cancer biomarkers (PSA, CEA, AFP) showed negative results by this method, validating the excellent specificity of the method. Then the assay was successfully applied to quantitatively detect CRP in peripheral blood plasma samples from lung cancer and breast cancer patients, and healthy people, facilitating the diagnosis of lung cancer. It holds a good prospect of POC protein biomarker detection.

Fluorescence-Converging Carbon Nanodots-Hybridized Silica Nanosphere.

Ultrabright carbon nanodots-hybridized silica nanospheres (CSNs) are synthesized through the Stöber process of silane functionalized C-dots. The fluorescence of carbon nanodots is converged intensely. A CSN is about 3800 times brighter than a single-carbon nanodot. Along with their high brightness and low cytotoxicity, CSNs also indicate their potential application in cellular labeling.

Rapid and Quantitative Detection of Avian Influenza A(H7N9) Virions in Complex Matrices Based on Combined Magnetic Capture and Quantum Dot Labeling

Avian influenza A(H7N9) virus, which emerged in China in the spring of 2013, has infected hundreds of people and resulted in many deaths. Herein, a rapid and quantitative assay is proposed for the one-step detection of H7N9 virions. Immunomagnetic nanospheres (IMNs) and antibody-conjugated quantum dots (Ab-QDs) are simultaneously employed to capture and identify the target virus, leading to a high efficiency, good specificity, and strong anti-interference ability. Moreover, this reliable detection assay, which combines the efficient magnetic enrichment and the unique photophysical properties of QDs, can achieve a high sensitivity for a low detection limit. At the same time, this detection strategy shows great flexibility for employment in a variety of fluorescence detectors, including fluorescence spectrometry, microscope assays, and handheld UV lamp tests. Furthermore, our one-step detection strategy induces very little change in the integrity of the vulnerable virions, which enables additional genotyping testing following the fluorescence detection. The present study, thus, reports a rapid and quantitative approach for the detection of H7N9 virions based on simultaneous magnetic capture and QD labeling, thereby providing a higher probability for detection and therefore faster diagnosis of H7N9-infected patients.

Biofunctionalized magnetic nanospheres-based cell sorting strategy for efficient isolation, detection and subtype analyses of heterogeneous circulating hepatocellular carcinoma cells.

Hepatocellular carcinoma (HCC) is an awful threat to human health. Early-stage HCC may be detected by isolation of circulating tumor cells (CTCs) from peripheral blood samples, which is beneficial to the diagnosis and therapy. However, the extreme rarity and high heterogeneity of HCC CTCs have been restricting the relevant research. To achieve an efficient isolation, reliable detection and subtype analyses of heterogeneous HCC CTCs, herein, we present a cell sorting strategy based on anti-CD45 antibody-modified magnetic nanospheres. By this strategy, leukocyte depletion efficiency was up to 99.9% within 30min in mimic clinical samples, and the purity of the spiked HCC cells was improved 265-317-fold. Besides, the isolated HCC cells remained viable at 92.3% and could be directly recultured. Moreover, coupling the convenient, fast and effective cell sorting strategy with specific ICC identification via biomarkers AFP and GPC3, HCC CTCs were detectable in peripheral blood samples, showing the potential for HCC CTC detection in clinic. Notably, this immunomagnetic cell sorting strategy enabled isolating more heterogeneous HCC cells compared with the established EpCAM-based methods, and further achieved characterization of three different CTC subtypes from one clinical HCC blood sample, which may assist clinical HCC analyses such as prognosis or personalized treatment.

Ultrasensitive Ebola Virus Detection Based on Electroluminescent Nanospheres and Immunomagnetic Separation

The 2014-16 Ebola virus (EBOV) outbreak in West Africa has attracted widespread concern. Rapid and sensitive detection methods are urgently needed for diagnosis and treatment of the disease. Here, we propose a novel method for EBOV detection based on efficient amplification of electroluminescent nanospheres (ENs) coupled with immunomagnetic separation. Uniform ENs are made by embedding abundant amounts of CdSe/ZnS quantum dots (QDs) into copolymer nanospheres through simple ultrasound. Compared to QDs, ENs can enhance electroluminescence (ECL) signals by approximately 85-fold, achieving a signal-to-background ratio high enough for EBOV detection. The introduction of magnetic nanobeads (MBs) can selectively separate targets from complex samples, simplifying the operation process and saving time. The presence of MBs can amplify ECL by approximately 3-fold, improving detection sensitivity. By integration of ENs with MBs, a sensitive electroluminescence biosensor is established for EBOV detection. The linear range is 0.02-30 ng/mL with a detection limit of 5.2 pg/mL. This method provides consistent reproducibility, specificity, and anti-interference ability and is highly promising in clinical diagnosis applications.

Efficient Enrichment and Analyses of Bacteria at Ultralow Concentration with Quick-Response Magnetic Nanospheres

Enrichment and purification of bacteria from complex matrices are crucial for their detection and investigation, in which magnetic separation techniques have recently show great application advantages. However, currently used magnetic particles all have their own limitations: Magnetic microparticles exhibit poor binding capacity with targets, while magnetic nanoparticles suffer slow magnetic response and high loss rate during treatment process. Herein, we used a highly controllable layer-by-layer assembly method to fabricate quick-response magnetic nanospheres (MNs), and with Salmonella typhimurium as a model, we successfully achieve their rapid and efficient enrichment. The MNs combined the advantages of magnetic microparticles and nanoparticles. On the one hand, the MNs had a fast magnetic response, and almost 100% of the MNs could be recovered by 1 min attraction with a simple magnetic scaffold. Hence, using antibody conjugated MNs (immunomagnetic nanospheres, IMNs) to capture bacteria hardly generated loss and did not need complex separation tools or techniques. On the other hand, the IMNs showed much excellent capture capacity. With 20 min interaction, almost all of the target bacteria could be captured, and even only one bacterium existing in the samples was not missed, comparing with the immunomagnetic microparticles which could only capture less than 50% of the bacteria. Besides, the IMNs could achieve the same efficient enrichment in complex matrices, such as milk, fetal bovine serum, and urine, demonstrating their good stability, strong anti-interference ability, and low nonspecific adsorption. In addition, the isolated bacteria could be directly used for culture, polymerase chain reaction (PCR) analyses, and fluorescence immunoassay without a release process, which suggested our IMNs-based enrichment strategy could be conveniently coupled with the downstream identification and analysis techniques. Thus, the MNs provided by this work showed great superiority in bacteria enrichment, which would be a promising tool for bacteria detection and investigation.

Cupric Ions Induce the Oxidation and Trigger the Aggregation of Human Superoxide Dismutase 1

Background Amyotrophic lateral sclerosis (ALS), partly caused by the mutations and aggregation of human copper, zinc superoxide dismutase (SOD1), is a fatal degenerative disease of motor neurons. Because SOD1 is a major copper-binding protein present at relatively high concentration in motor neurons and copper can be a harmful pro-oxidant, we want to know whether aberrant copper biochemistry could underlie ALS pathogenesis. In this study, we have investigated and compared the effects of cupric ions on the aggregation of ALS-associated SOD1 mutant A4V and oxidized wild-type SOD1. Methodology/Principal Findings As revealed by 90° light scattering, dynamic light scattering, SDS-PAGE, and atomic force microscopy, free cupric ions in solution not only induce the oxidation of either apo A4V or Zn2-A4V and trigger the oligomerization and aggregation of oxidized A4V under copper-mediated oxidative conditions, but also trigger the aggregation of non-oxidized form of such a pathogenic mutant. As evidenced by mass spectrometry and SDS-PAGE, Cys-111 is a primary target for oxidative modification of pathological human SOD1 mutant A4V by either excess Cu2+ or hydrogen peroxide. The results from isothermal titration calorimetry show that A4V possesses two sets of independent binding sites for Cu2+: a moderate-affinity site (106 M-1) and a high-affinity site (108 M-1). Furthermore, Cu2+ binds to wild-type SOD1 oxidized by hydrogen peroxide in a way similar to A4V, triggering the aggregation of such an oxidized form. Conclusions/Significance We demonstrate that excess cupric ions induce the oxidation and trigger the aggregation of A4V SOD1, and suggest that Cu2+ plays a key role in the mechanism of aggregation of both A4V and oxidized wild-type SOD1. A plausible model for how pathological SOD1 mutants aggregate in ALS-affected motor neurons with the disruption of copper homeostasis has been provided.