Guohua Zhou

China Pharmaceutical University, Nan-ching-hsü, Jiangxi Sheng, China

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Publications (45)160.7 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Colorimetric DNA detection is preferable to methods in clinical molecular diagnostics, because no expensive equipment is required. Although many gold nanoparticle-based colorimetric DNA detection strategies have been developed to analyze DNA sequences of interest, few of them can detect somatic mutations due to their insufficient specificity. In this study, we proposed a colorimetric DNA detection method by coupling invasive reaction with nicking endonuclease-assisted nanoparticles amplification (IR-NEANA). A target DNA firstly produces many flaps by invasive reaction. Then the flaps are converted to targets of nicking reaction-assisted nanoparticles amplification by ligation reaction to produce the color change of AuNPs, which can be observed by naked eyes. The detection limit of IR-NEANA was determined as 1 pM. Most importantly, the specificity of the method is high enough to pick up as low as 1% mutant from a large amount of wild-type DNA backgrounds. The EGFR gene mutated at c.2573 T>G in 9 tissue samples from non-small cell lung cancer patients were successfully detected by using IR-NEANA, suggesting that our proposed method can be used to detect somatic mutations in biological samples.
    Biosensors & Bioelectronics 04/2015; 66. · 6.45 Impact Factor
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    ABSTRACT: The high background signal caused by non-specific amplification (NSA) is a serious issue when using the conventional exponential amplification reaction (EXPAR). We describe a novel method of suppressing NSA using 10 mg L−1 graphene oxide (GO) to prevent non-specific binding of DNA polymerase to the template, resulting in ultra-low background. A side effect of the use of GO is the slow release of ssDNA from the GO surface, with the positive signal decreasing accordingly. The problem of low signal intensity is addressed by applying a 2 μM solution of single-stranded binding protein (SSB) to accelerate the release of template for EXPAR. The use of GO and SSB in EXPAR can substantially suppress NSA, but it does not compromise the performance of the quantification step. The improved EXPAR presented here can detect concentration as low as 5 aM of trigger, which is approximately four orders of magnitude lower than that of conventional EXPAR under the same experimental conditions. Graphical Abstract A high background in exponential amplification reaction (EXPAR) is effectively suppressed by using graphene oxide (GO). The slow release of ssDNA from GO surface is countered by using single-stranded binding protein. This improved EXPAR can detect as little as 5 aM of a trigger.
    Microchimica Acta 12/2014; · 3.43 Impact Factor
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    ABSTRACT: A novel DNA detection assay was proposed by invasive reaction coupled with molecular beacon assisted strand-displacement signal amplification (IRASA). Target DNAs are firstly hybridized to two probes to initiate invasive reaction to produce amplified flaps. Then these flaps are further amplified by strand-displacement signal amplification.The detection limit was around 0.2 pM.
    Chemical Communications 09/2014; · 6.38 Impact Factor
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    ABSTRACT: Pyrosequencing is a powerful tool widely used in genetic analysis, however template preparation prior to pyrosequencing is still costly and time-consuming. To achieve an inexpensive and labor-saving template preparation for pyrosequencing, we have successfully developed a single-tube multiplex PCR including a pre-amplification and a universal amplification. In the process of pre-amplification, a low concentration of target-specific primers tagged with universal ends introduced universal priming regions into amplicons. In the process of universal amplification, a high concentration of universal primers was used for yielding amplicons with various SNPs of interest. As only a universal biotinylated primer and one step of single-stranded DNA preparation were required for typing multiple SNPs located on different sequences, pyrosequencing-based genotyping became time-saving, labor-saving, sample-saving, and cost-saving. By a simple optimization of multiplex PCR condition, only a 4-plex and a 3-plex PCR were required for typing 7 SNPs related to tamoxifen metabolism. Further study showed that pyrosequencing coupled with an improved multiplex PCR protocol allowed around 30% decrease of either typing cost or typing labor. Considering the biotinylated primer and the optimized condition of the multiplex PCR are independent of SNP locus, it is easy to use the same condition and the identical biotinylated primer for typing other SNPs. The preliminary typing results of the 7 SNPs in 11 samples demonstrated that multiplex PCR-based pyrosequencing could be promising in personalized medicine at a low cost.
    Journal of Nanoscience and Nanotechnology 06/2014; 14(6):4363-70. · 1.15 Impact Factor
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    ABSTRACT: The aim of this study was to evaluate (+)-catechin and (-)-epigallocatechin gallate (EGCG) cellular uptake and transport across human intestinal Caco-2 cell monolayer in both the absence and presence of niosomal carrier in variable conditions. The effect of free drugs and drug-loaded niosomes on the growth of Caco-2 cells was studied. The effects of time, temperature, and concentration on drug cellular uptake in the absence or presence of its niosomal delivery systems were investigated. The intestinal epithelial membrane transport of the drug-loaded niosomes was examined using the monolayer of the human Caco-2 cells. The kinetics of transport, and the effect of temperature, adenosine triphosphate inhibitor, permeability glycoprotein inhibitor, multidrug resistance-associated protein 2 inhibitor, and the absorption enhancer on transport mechanism were investigated. It was found that the uptake of catechin, EGCG, and their niosomes by Caco-2 cells was 1.22±0.16, 0.90±0.14, 3.25±0.37, and 1.92±0.22 μg/mg protein, respectively (n=3). The apparent permeability coefficient values of catechin, EGCG, and their niosomes were 1.68±0.16, 0.88±0.09, 2.39±0.31, and 1.42±0.24 cm/second (n=3) at 37°C, respectively. The transport was temperature- and energy-dependent. The inhibitors of permeability glycoprotein and multidrug resistance-associated protein 2 and the absorption enhancer significantly enhanced the uptake amount. Compared with the free drugs, niosomal formulation significantly enhanced drug absorption. Additionally, drug-loaded niosomes exhibited stronger stability and lower toxicity. These findings showed that the oral absorption of tea flavonoids could be improved by using the novel drug delivery systems.
    International Journal of Nanomedicine 01/2014; 9:2157-65. · 4.20 Impact Factor
  • Qinxin Song, Guijiang Wei, Guohua Zhou
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    ABSTRACT: A portable bioluminescence analyser for detecting the DNA sequence of genetically modified organisms (GMOs) was developed by using a photodiode (PD) array. Pyrosequencing on eight genes (zSSIIb, Bt11 and Bt176 gene of genetically modified maize; Lectin, 35S-CTP4, CP4EPSPS, CaMV35S promoter and NOS terminator of the genetically modified Roundup ready soya) was successfully detected with this instrument. The corresponding limit of detection (LOD) was 0.01% with 35 PCR cycles. The maize and soya available from three different provenances in China were detected. The results indicate that pyrosequencing using the small size of the detector is a simple, inexpensive, and reliable way in a farm/field test of GMO analysis.
    Food Chemistry 01/2014; 154:78–83. · 3.33 Impact Factor
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    ABSTRACT: Nucleic acid analysis in a single cell is very important, but the extremely small amount of template in a single cell requires a detection method more sensitive than the conventional method. In this paper, we describe a novel assay allowing a single cell genotyping by coupling improved linear-after-the-exponential-PCR (imLATE-PCR) on a modified glass slide with highly sensitive pyrosequencing. Due to the significantly increased yield of ssDNA in imLATE-PCR amplicons, it is possible to employ pyrosequencing to sequence the products from 1 μL chip PCR which directly used a single cell as the starting material. As a proof-of-concept, the 1555A>G mutation (related to inherited deafness) on mitochondrial DNA and the SNP 2731C>T of the BRCA1 gene on genomic DNA from a single cell were successfully detected, indicating that our single-cell-pyrosequencing method has high sensitivity, simple operation and is low cost. The approach has promise to be of efficient usage in the fields of diagnosis of genetic disease from a single cell, for example, preimplantation genetic diagnosis (PGD).
    The Analyst 07/2013; · 4.23 Impact Factor
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    ABSTRACT: LAMP is an isothermal amplification method that can achieve ultra-high sensitivity and specificity. However, the conventional detection of LAMP amplicons can lead to cross-contamination due to the need to open the reaction tube which contains a large number of amplicons. To achieve closed-tube LAMP detection, we have developed a method that separates a solution of SYBR Green I (SGI) from the LAMP reagents using temperature-sensitive wax. The SGI is sealed in the bottom of the tube so not to interfere with the LAMP reaction, but is released into the mixture after the completion of the reaction by melting the wax. To enable the analysis of the closed-tube LAMP samples automatically, an instrument based on this new method was constructed. The background measurement of the LAMP due to primer dimers was significantly reduced by detecting the amplicons at 75 degrees C. HBV and 2009 H1N1 virus were successfully analyzed by the LAMP assay using tubes containing wax-sealed SGI and the prototype instrument, indicating that the method has the advantage of easy set-up (no extra components need to be added into the LAMP mixture for detection), high sensitivity (fluorescent intercalator), low background (detected at 75 degrees C) and no cross-contamination (closed-tube). Therefore, the novel LAMP detection, coupled with the instrument has the potential to be a diagnostic tool for a number of clinical applications in hospitals as well as on-site screening of pathogenic agents.
    Journal of Nanoscience and Nanotechnology 06/2013; 13(6):3999-4005. · 1.15 Impact Factor
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    ABSTRACT: A novel assay based on a solvent for inducing the aggregation of AuNPs colloid was proposed to discriminate ssDNA from dsDNA. Eleven organic solvents with different polarity were investigated, and it was found that DMSO was possible to aggregate AuNPs at the amount of only 0.4 microL in a 50-microL detection system. Further research showed that 0.8 microL of DMSO could discriminate the ssDNA from dsDNA. Colorimetric detection with various conditions, including the ratio of the target to the probe, and the concentration of AuNPs and DNA, was investigated. The proposed method was successfully used for SNP typing, and unambiguous discrimination of a wild type from a mutant was obtained for the templates with the mismatched base at the 5'-end or in the middle of the target sequence. As no requirement of gold modification and detection instrument, we believe that this method will be much low in the cost for DNA detection.
    Journal of Nanoscience and Nanotechnology 06/2013; 13(6):3805-9. · 1.15 Impact Factor
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    ABSTRACT: Allelic ratio of an SNP has been used for prenatal diagnosis of fetal trisomy 21 by MALDI-TOF mass spectrometry (MS). Because MALDI-TOF MS is challenging in quantification performance, pyrosequencing was proposed to replace MS by better quantification of allelic ratios. To achieve a simple and rapid clinical diagnostic, PCR with "HpH Buffer" (a buffer with a high pH) was developed to directly amplify amniotic fluid. By the established assay, 114 samples of amniotic fluid were analyzed by pyrosequencing five SNPs of each sample; the allelic ratios of euploid heterozygotes were thus calculated to determine the cut-off values for prenatal diagnosis of trisomy 21. The panel of five SNPs were high in heterozygosity so that at least one heterozygote was found in each sample, and 86% of the samples had at least two heterozygotes, giving a nearly 100% sensitivity (population coverage) of the assay. By using the cut-off values of each SNP, 20 pre-diagnosed clinical samples were detected as trisomy 21 carriers with a confidence level over 99%, indicating that our method and karyotyping analysis were consistent in results. In conclusion, this pyrosequencing-based approach, coupled with direct amplification of amniotic fluid, is accurate in quantitative genotyping and simple in operation. We believe that the approach could be a promising alternative to karyotyping analysis in prenatal diagnosis.
    The Analyst 03/2013; · 4.23 Impact Factor
  • Bingjie Zou, Yinjiao Ma, Guohua Zhou
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    ABSTRACT: Although many approaches based on template replication were developed and applied in DNA detection, cross-contamination from amplicons is always a vexing problem. Thus, signal amplification is preferable for DNA detection due to its low risk of cross-contamination from amplicons. Here, we proposed a cascade enzymatic signal amplification (termed as CESA) by coupling Afu flap endonuclease with nicking endonuclease, including three steps: invasive signal amplification, flap ligation, and nicking endonuclease signal amplification. Because of the advantages of low risk of contamination, no sequence requirement of target DNA, and the universal reaction conditions for any target detection, CESA has a great potential in clinical diagnosis.
    Methods in molecular biology (Clifton, N.J.) 01/2013; 1039:131-7. · 1.29 Impact Factor
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    ABSTRACT: Quantitative analysis of virus nucleic acids is essential for monitoring the efficacy of medical treatment based on the copy numbers of virus's RNA or DNA in blood. To quantitatively detect virus nucleic acids in blood, here an internal amplification control (IAC) coupled with a nanoparticle-based DNA biosensor was proposed. The IACs with a specific sequence were designed and spiked into serum before nucleic acids extraction. Sequences of the IACs and the targets only differ in the base order of one PCR priming site; thus, the IACs and the targets are identical in Tm, giving the same amplification efficiency during PCR. To visually detect amplicons, a dipstick biosensor based on streptavidin-functionalized nanoparticles is employed. By comparing color densities of a test zone with an IAC zone on the biosensor, the content of the target in serum can be semi-quantitatively analyzed. This approach has achieved the detection of HBV DNA at approximately 100 copies of the pathogen load. The feasibility of this method is demonstrated by successful semi-quantification of pathogen load in 30 clinical samples from HBV-infected patients. These data indicate that the introduction of an IAC and nanoparticle-based dipstick-type biosensor could be a powerful tool in point of care testing (POCT).
    Biosensors & Bioelectronics 11/2012; 42C:261-266. · 6.45 Impact Factor
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    ABSTRACT: A 3-dimensional (3-D) polyacrylamide gel microarray based on dual-color fluorescence hybridization was an efficient SNP typing method with a high-throughput, but it is expensive to use dual dye-labeled allele-specific probes to type various SNPs. To lower the typing cost on 3-D polyacrylamide gel microarray, we propose a novel method by incorporating Cy5-dCTP into label-free allele-specific probes hybridizing to gel-immobilized targets. The method is much simple. At first, raw PCR products without any purification was spotted on the acryl-modified slides to copolymerize with acrylamide monomers. Then a pair of allele-specific probes were respectively added into two different areas of a hydrogel chip to hybridize with the single-stranded DNA targets immobilized in the gel-pads. Before extension reaction with Cy5-dCTP, electrophoresis was performed on the gel chip to remove non-specific allele-specific probes, and a high specificity was thus obtained. After the extension reaction, electrophoresis was used once more to remove the unincorporated Cy5-dCTP absorbed in the gel pads, and a low background image was achieved. The method was successfully employed to type the SNP (C14417G) in the OLR-1 gene for 40 different samples, and the typing results were consistent with those by pyrosequencing, indicating that the proposed method is accurate and specific in SNP typing. As no use of dye-modified probes, the typing cost is significantly decreased in comparison with the conventional typing method based on dual-color fluorescence hybridization, in particular when typing multiple SNPs. In addition to the low cost, our method has a low risk of cross-contamination from PCR amplicons due to no need of purification step of PCR products. Although only proof-of-concept results were given, we believe that the proposed method should be very useful for screening the biomarkers related to disease-susceptibility and personalized medicine where detection of many SNPs in different genes is required.
    Journal of Nanoscience and Nanotechnology 09/2012; 12(9):6887-92. · 1.15 Impact Factor
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    ABSTRACT: Somatic mutations in stool DNA are quite specific to colorectal cancer (CRC), but a method being able to detect the extraordinarily low amounts of mutants is challengeable in sensitivity. We proposed a hydrogel bead-array to digitally count CRC-specific mutants in stool at a low cost. At first, multiplex amplification of targets containing multiple mutation loci of interest is carried out by a target enriched multiplex PCR (Tem-PCR), yielding the templates qualified for emulsion PCR (emPCR). Then, after immobilizing the beads from emPCR on a glass surface, the incorporation of Cy3-dUTP into the mutant-specific probes, which are specifically hybridized with the amplified beads from emPCR, is used to color the beads coated with mutants. As all amplified beads are hybridized with the Cy5-labeled universal probe, a mutation rate is readily obtained by digitally counting the beads with different colors (yellow and red). A high specificity of the method is achieved by removing the mismatched probes in a bead-array with electrophoresis. The approach has been used to simultaneously detect 8 mutation loci within the APC, TP53, and KRAS genes in stools from eight CRC patients, and 50% of CRC patients were positively diagnosed; therefore, our method can be a potential tool for the noninvasive diagnosis of CRC.
    Analytical Chemistry 06/2012; 84(13):5645-52. · 5.82 Impact Factor
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    ABSTRACT: Pyrosequencing is a tool based on bioluminescence reaction for real-time analyzing DNA sequences. The sensitivity of pyrosequencing mainly depends on luciferase in reaction mixture. However, the instability of pyrosequencing reagents caused by fragile wild Photinus pyralis luciferase (PpL) in conventional pyrosequencing usually leads to unsatisfied results, which limits the application of pyrosequencing. In order to improve the stability of pyrosequencing reagents, the coding sequences of mutant thermostable Luciola lateralis luciferase (rt-LlL) was synthesized, and inserted into the plasmid of pET28a(+) to express the thermostable rt-LlL with a 6 x His-tag in the N terminal. The purified rt-LlL with the molecular mass of 60 kDa was obtained by Ni-affinity chromatography. The specific activity of rt-LlL was determined as 4.29 x 10(10) RLU/mg. Moreover, the thermostability of rt-LlL was investigated, and the results showed that rt-LlL had activity at 50 degrees C, and remained 90% of activity after incubated at 40 degrees C for 25 min. Finally, rt-LlL was used to substitute commercial Photinus pyralis luciferase in conventional pyrosequencing reagent to get thermostable pyrosequencing reagent. Comparing with conventional pyrosequencing reagent, the thermostable pyrosequencing reagent is more stable, and it's activity would not lose when incubated at 37 degrees C for 1 h. This study laid foundation of establishing reliable and stable pyrosequencing system which would be applied in Point-of-Care Testing.
    Sheng wu gong cheng xue bao = Chinese journal of biotechnology 06/2012; 28(6):763-71.
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    ABSTRACT: The loop-mediated isothermal amplification (LAMP) is a well-developed method for replicating a targeted DNA sequence with a high specificity, but multiplex LAMP detection is difficult because LAMP amplicons are very complicated in structure. To allow simultaneous detection of multiple LAMP products, a series of target-specific barcodes were designed and tagged in LAMP amplicons by FIP primers. The targeted barcodes were decoded by pyrosequencing on nicked LAMP amplicons. To enable the nicking reaction to occur just near the barcode regions, the recognition sequence of the nicking endonuclease (NEase) was also introduced into the FIP primer. After the nicking reaction, pyrosequencing started at the nicked 3' end when the added deoxyribonucleoside triphosphate (dNTP) was complementary to the non-nicked strand. To efficiently encode multiple targets, the barcodes were designed with a reporter base and two stuffer bases, so that the decoding of a target-specific barcode only required a single peak in a pyrogram. We have successfully detected the four kinds of pathogens including hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), and Treponema pallidum (TP), which are easily infected in blood, by a 4-plex LAMP in a single tube, indicating that barcoded LAMP coupled with NEase-mediated pyrosequencing is a simple, rapid, and reliable way in multiple target identification.
    Analytical Chemistry 03/2012; 84(8):3758-63. · 5.82 Impact Factor
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    ABSTRACT: A novel dye-free labeling method for a multiplex bioassay was proposed by using short sequence-based barcodes consisting of a reporter base and repeats of two stuffer bases; then, the barcodes were quantitatively decoded by a single pyrosequencing assay without any pre-separation.
    Chemical Communications 02/2012; 48(18):2445-7. · 6.38 Impact Factor
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    ABSTRACT: Pyrosequencing is an ideal tool for verifying the sequence of amplicons. To enable pyrosequencing on amplicons from nucleic acid sequence-based amplification (NASBA), asymmetric NASBA with unequal concentrations of T7 promoter primer and reverse transcription primer was proposed. By optimizing the ratio of two primers and the concentration of dNTPs and NTPs, the amount of single-stranded cDNA in the amplicons from asymmetric NASBA was found increased 12 times more than the conventional NASBA through the real-time detection of a molecular beacon specific to cDNA of interest. More than 20 bases have been successfully detected by pyrosequencing on amplicons from asymmetric NASBA using Human parainfluenza virus (HPIV) as an amplification template. The primary results indicate that the combination of NASBA with a pyrosequencing system is practical, and should open a new field in clinical diagnosis.
    The Analyst 12/2011; 136(24):5229-33. · 4.23 Impact Factor
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    ABSTRACT: Detection of nucleic acids with signal amplification is preferable in clinical diagnosis. A novel approach was developed for signal amplification by coupling invasive reaction with hyperbranched rolling circle amplification (HRCA). Invasive reaction, which does not rely on specific recognition sequences in a target but a specific structure formed by the specific binding of an upstream probe and a downstream probe to a target DNA, can generate thousands of flaps from one target DNA; then the flaps are ligated with padlock probes to form circles, which are the templates of HRCA. As HRCA amplicon sequence is free of target DNA sequence, signal amplification is achieved. Because flap sequence is the same to any target of interest, HRCA is universal; the detection cost is hence greatly reduced. The sensitivity of the proposed method is less than 1 fM artificial DNA targets; and the specificity of the method is high enough to discriminate one base difference in the target sequence. The feasibility was verified by detecting real biological samples from HBV carriers, indicating that the method is highly sensitive, cost-effective, and has a low risk of cross-contamination from amplicons. These properties should give great potential in clinical diagnosis.
    The Analyst 12/2011; 137(3):729-34. · 4.23 Impact Factor
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    ABSTRACT: Streptococcus suis (S. suis for short) can cause a variety of infections in pigs, and the infections have brought about great losses in the swine industry and some cases of deaths in human beings. In order to rapidly diagnose and control the infections of S. suis, we designed a pyrosequencing-based assay to identify the serotypes of S. suis. In the assay, pyrosequencing is used to genotype most of the pathogenic serotypes of S. suis by detecting five informative regions on the Chaperonin 60 (cpn60) gene and one species-specific region on the 16S rRNA gene, and further a few undistinguished serotypes by pyrosequencing were finely discriminated by multiplex PCR of serotype-specific fragments on the cps gene as well as species-specific fragments on the 16S rRNA gene. Through carefully designing the dispensing order of dNTP for each pyrosequencing reaction, the serotypes of S. suis could be discriminated by four pyrosequencing reactions within three hours. Five reference serotypes and three clinical strains were successfully detected and genotyped by our assay. The results indicated that our assay is a reliable, information-rich diagnostic method for the accurate detection ofS. suis serotypes.
    Analytical methods 11/2011; 3(11):2517-2523. · 1.86 Impact Factor

Publication Stats

109 Citations
160.70 Total Impact Points

Institutions

  • 2008–2014
    • China Pharmaceutical University
      • • School of Pharmacy
      • • School of Life Science and Technology
      • • Division of Analytical Chemistry
      Nan-ching-hsü, Jiangxi Sheng, China
  • 2012–2013
    • Nanjing Medical University
      • Department of Pharmacology
      Nanjing, Jiangsu Sheng, China
  • 2009
    • Soochow University (PRC)
      • Institute of Pharmaceutical Analysis
      Suzhou, Jiangsu Sheng, China
  • 2006
    • Southeast University (China)
      Nan-ching-hsü, Jiangxi Sheng, China