Cong-Ying Wen

Fluorescent-magnetic-biotargeting multifunctional nanobioprobes for detecting and isolating multiple types of tumor cells.

Fluorescent-magnetic-biotargeting multifunctional nanobioprobes (FMBMNs) have attracted great attention in recent years due to their increasing, important applications in biomedical research, clinical diagnosis, and biomedicine. We have previously developed such nanobioprobes for the detection and isolation of a single kind of tumor cells. Detection and isolation of multiple tumor markers or tumor cells from complex samples sensitively and with high efficiency is critical for the early diagnosis of tumors, especially malignant tumors or cancers, which will improve clinical diagnosis outcomes and help to select effective treatment approaches. Here, we expanded the application of the monoclonal antibody (mAb)-coupled FMBMNs for multiplexed assays. Multiple types of cancer cells, such as leukemia cells and prostate cancer cells, were detected and collected from mixed samples within 25 min by using a magnet and an ordinary fluorescence microscope. The capture efficiencies of mAb-coupled FMBMNs for the above-mentioned two types of cells were 96% and 97%, respectively. Furthermore, by using the mAb-coupled FMBMNs, specific and sensitive detection and rapid separation of a small number of spiked leukemia cells and prostate cancer cells in a large population of cultured normal cells (about 0.01% were tumor cells) were achieved simply and inexpensively without any sample pretreatment before cell analysis. Therefore, mAb-coupled multicolor FMBMNs may be used for very sensitive detection and rapid isolation of multiple cancer cells in biomedical research and medical diagnostics.

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.

One-Step Sensitive Detection of Salmonella typhimurium by Coupling Magnetic Capture and Fluorescence Identification with Functional Nanospheres

Sensitive, rapid, and reliable detection of bacteria has always been pursued due to the great threat of the bacteria to human health. In this study, a convenient one-step strategy for detecting Salmonella typhimurium was developed. Immunomagnetic nanospheres (IMNS) and immunofluorescent nanospheres (IFNS) were used to specifically capture and recognize S. typhimurium simultaneously. After magnetic separation, the sandwich immune complexes (IMNS-bacteria-IFNS) were detected under a fluorescence microscope with a detection limit as low as ca. 10 CFU/mL. When they were detected by fluorescence spectrometer, a linear range was exhibited at the concentration from 10(5) to 10(7) CFU/mL with R(2) = 0.9994. Compared with the two-step detection strategy, in which the bacteria were first captured with the IMNS and subsequently identified with the IFNS, this one-step strategy simplified the detection process and improved the sensitivity. Escherichia coli and Shigella flexneri both showed negative results with this method, indicating that this method had excellent selectivity and specificity. Moreover, this method had strong anti-interference ability, and it had been successfully used to detect S. typhimurium in synthetic samples (milk, fetal bovine serum, and urine), showing the potential application in practice.

A multicomponent recognition and separation system established via fluorescent, magnetic, dualencoded multifunctional bioprobes

Accurate and rapid recognition and separation of multiple types of biological targets such as molecules, cells, bacteria or viruses from complex sample mixtures is of great importance for a wide range of diagnostic and therapeutic strategies. To achieve this goal, a set of fluorescent, magnetic, dual-encoded multifunctional bioprobes has been constructed by co-embedding different-sized quantum dots and varying amounts of γ-Fe(2)O(3) magnetic nanoparticles into swollen poly(styrene/acrylamide) copolymer nanospheres. The dual-encoded bioprobes, which possessed different photoluminescent property and magnetic susceptibility, were proven to be capable of simultaneously recognizing and separating multiple components from a complex sample when three kinds of lectins were used as the targets. The lectins were separated with high efficiency and kept their bioactivity during the process. Compared to the conventional batchwise separation, this method does not require a large number of sequential reaction steps, which is economical of time and can be very reagent-saving. By combining the multiplexing capability of quantum dots with the superparamagnetic properties of iron oxide nanoparticles, this dual-encoded technique is expected to open new opportunities in high-throughput and multiplex bioassays, such as cell sorting, proteomical and genomical applications, drug screening etc.

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.

Fluorescent-magnetic dual-encoded nanospheres: a promising tool for fast-simultaneous-addressable high-throughput analysis

Bead-based optical encoding or magnetic encoding techniques are promising in high-throughput multiplexed detection and separation of numerous species under complicated conditions. Therefore, a self-assembly strategy implemented in an organic solvent is put forward to fabricate fluorescent-magnetic dual-encoded nanospheres. Briefly, hydrophobic trioctylphosphine oxide-capped CdSe/ZnS quantum dots (QDs) and oleic acid-capped nano-γ-Fe2O3 magnetic particles are directly, selectively and controllably assembled on branched poly(ethylene imine)-coated nanospheres without any pretreatment, which is crucial to keep the high quantum yield of QDs and good dispersibility of γ-Fe2O3. Owing to the tunability of coating amounts of QDs and γ-Fe2O3 as well as controllable fluorescent emissions of deposited-QDs, dual-encoded nanospheres with different photoluminescent emissions and gradient magnetic susceptibility are constructed. Using this improved layer-by-layer self-assembly approach, deposition of hydrophobic nanoparticles onto hydrophilic carriers in organic media can be easily realized; meanwhile, fluorescent-magnetic dual-functional nanospheres can be further equipped with readable optical and magnetic addresses. The resultant fluorescent-magnetic dual-encoded nanospheres possess both the unique optical properties of QDs and the superparamagnetic properties of γ-Fe2O3, exhibiting good monodispersibility, huge encoding capacity and nanoscale particle size. Compared with the encoded microbeads reported by others, the nanometre scale of the dual-encoded nanospheres gives them minimum steric hindrance and higher flexibility.

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.

Control of magnetic field distribution by using nickel powder@PDMS pillars in microchannels

A simple and robust approach to control the magnetic field distribution by nickel powder@PDMS pillars was established. The nickel powder@PDMS pillars were fabricated in several simple steps, using a simple and robust method, and no training in techniques or expensive equipment is necessary compared to other methods. The localized magnetic field distributions in microchannels can be tailored by the nickel powder@PDMS pillars with automatic generation of high magnetic field gradients around them due to the high relative magnetic permeability of the nickel powder@PDMS. The numerical simulation and red fluorescent magnetic nanoparticle capture experiment results convinced us that our approach could effectively control the localized magnetic field distribution in the microchannels. Two kinds of tailoring events were studied at the powder@PDMS pillars in the microchannels underneath two different external magnetic fields. To the best of our knowledge, this is the first time different localized magnetic field distributions have been obtained in microchannels by nickel powder@PDMS pillars due to different external magnetic fields. This approach was used to capture fluorescent magnetic nanoparticles and magnetic bead–yeast cell complexes. We believe that this approach has great potential applications in chemistry, biology, biomedicine and tissue engineering.