Jiaxin Zheng

Multiplex immunodetection of tumor markers with a suspension array built upon core–shell structured functional fluorescence-encoded microspheres

A new suspension array built upon laboratory-prepared functional fluorescence-encoded polystyrene beads (FFPBs) was developed for multiplex immunodetection of tumor markers. The FFPBs were synthesized by copolymerizing rhodamine 6G (R6G) and carboxyl function groups on the surface of the seed beads forming a core-shell structure. The fabrication process was facile and the encoding fluorescence intensity of the beads can be precisely controlled by adjusting the quantity of R6G. In present work, we demonstrated that the quantity variation of impregnated R6G had negligible effect on the coupling efficiency of biomolecules onto the surface of the FFPBs. The R6G encoding fluorescence remained good monodispersity upon capture probe coupling and immunocomplex formation. No fluorescence resonance energy transfer was observed between the R6G doped in the bead shell and fluorophore used for antibody labeling. Under the optimal conditions, the proposed suspension array allowed simultaneous detection of alpha-fetoprotein, carcinoembryonic antigen, and prostate specific antigen in the ranges of 0.07-500 ng mL(-1), 1-2000 ng mL(-1), and 0.5-500 ng mL(-1), respectively, with detection limits of 0.0626 ng mL(-1), 0.554 ng mL(-1), and 0.250 ng mL(-1). Test on clinical serum samples demonstrated that the results obtained with suspension array were in good agreement with those of the reference electrochemiluminescence immunoassay method. We conclude that the laboratory-made FFPBs are sufficient as the microcarrier for the construction of suspension array in clinical diagnosis.

Liquid chromatography-mass spectrometry based serum peptidomic approach for renal clear cell carcinoma diagnosis.

Serum peptidomic approach was applied to investigate the peptidomic signature and discover the clinical biomarkers and biomarker patterns for RCC patients. The holistic orthogonal partial least-squares-discriminant analysis (OPLS-DA) based on qualified profile data successfully classified RCC patients from healthy controls, showing 100% sensitivity and specificity. Following critical criteria, several peptides presenting significant differences in serum level were picked out. The unsupervised hierarchical cluster analysis on those peptides was performed, showing 100% sensitivity and 93.3% specificity for RCC diagnosis regarding the present samples. Besides, receiver-operating characteristic (ROC) analysis was applied on single peptide biomarkers, with four peptides showing excellent predictive power. Among them, IYQLNSKLV and AGISMRSGDSPQD are reported for the first time for cancer detection.

Probing minority population of antibiotic-resistant bacteria.

The evolution and spread of antibiotic-resistant pathogens has become a major threat to public health. Advanced tools are urgently needed to quickly diagnose antibiotic-resistant infections to initiate appropriate treatment. Here we report the development of a highly sensitive flow cytometric method to probe minority population of antibiotic-resistant bacteria via single cell detection. Monoclonal antibody against TEM-1 β-lactamase and Alexa Fluor 488-conjugated secondary antibody were used to selectively label resistant bacteria green, and nucleic acid dye SYTO 62 was used to stain all the bacteria red. A laboratory-built high sensitivity flow cytometer (HSFCM) was applied to simultaneously detect the side scatter and dual-color fluorescence signals of single bacteria. By using E. coli JM109/pUC19 and E. coli JM109 as the model systems for antibiotic-resistant and antibiotic-susceptible bacteria, respectively, as low as 0.1% of antibiotic-resistant bacteria were accurately quantified. By monitoring the dynamic population change of a bacterial culture with the administration of antibiotics, we confirmed that under the antimicrobial pressure, the original low population of antibiotic-resistant bacteria outcompeted susceptible strains and became the dominant population after 5hours of growth. Detection of antibiotic-resistant infection in clinical urine samples was achieved without cultivation, and the bacterial load of susceptible and resistant strains can be faithfully quantified. Overall, the HSFCM-based quantitative method provides a powerful tool for the fundamental studies of antibiotic resistance and holds the potential to provide rapid and precise guidance in clinical therapies.

A Study of Human Bladder Cancer by Serum and Urine Metabonomics

National Science Foundation for Fostering Talents in Basic Research of the National Natural Science Foundation of China [J1030415]; Fujian Province Department of Science & Technology, China [2009D023]

Early diagnosis of urinary lithiasis via elementary profile of serum samples

An elemental analysis method was established to monitor the element levels in serum samples of 38 healthy controls and 38 stone patients. Based on the optimized platform combined with multivariate analysis, satisfactory results can be obtained for urinary lithiasis diagnosis with the concentrations of 9 elements, in which Ba, Ga, Sb, and Na are the top 4 elements of statistical significance. The patients could be subdivided into calcareous and non-calcareous stone patients by metallomic profiling; and it is found that Se plays the major role in this classification. This study indicates that serum elementary analysis gives an insight into the possibility of diagnosis of urinary lithiasis, subsequently it may allow estimation of the content subtype of stones. By means of this simple method of elemental profiling in serum, it might allow early prognosis and treatment guide to urinary lithiasis.

Association of polymorphisms in AGTR1 and AGTR2 genes with primary aldosteronism in the Chinese Han population

Hypothesis: Polymorphisms in angiotensin II type-1/2 receptor genes (AGTR1/AGTR2) may be involved in the pathogenesis of primary aldosteronism. The present study aims to reveal some loci susceptible to the disease on the genes in a group of Chinese Han nationality. Materials and methods: A case-control study was conducted in 202 patients and 188 controls. Ten tagging SNPs on AGTR1/AGTR2 were genotyped for all subjects via the method of multiplex PCR-ligase detection reaction. Statistical analysis was performed with chi-square test and logistic regression analysis. Results: rs3772616 on the AGTR1 gene was a factor for susceptibility to primary aldosteronism (p<0.001), and the TT genotype significantly decreased the risk of primary aldosteronism compared with the CC homozygote (p=0.008, adjusted OR=0.13; 95%CI: 0.03–0.59). The rs3772616 polymorphism was associated with primary aldosteronism under the additive and dominant models. The female carriers of the G allele in rs5193 showed a significant difference compared with the T allele. Conclusions: The AGTR1 rs3772616 polymorphism can be considered as a hereditary marker for primary aldosteronism, and in the Chinese Han population the rs5193 G allele seems to predispose to it only in women.

Detection of urinary trace elements and pattern recognition analysis in patients with renal cell carcinoma by inductively coupled plasma mass spectrometry

Objective: This study aims to observe the changes and diagnostic values of urinary trace elements in patients with renal cell carcinoma (RCC). Methods: A total of 28 RCC patients that had been performed radical surgery for more than 3 years were included into this case–control study; meanwhile, thirty healthy volunteers without kidney diseases were set as the control group over the same period. The levels of various urinary trace elements in both groups were measured, and the results were performed the significance analysis and pattern recognition analysis using with partial least square discriminant analysis and Fisher analysis. Results: The significance analysis showed that compared with the control group, the case group exhibited significantly reduced levels of Mg (26.3 mg/L for the case group vs. 52.1 mg/L for the control group, P < 0.05), V (72.9 μg/L for the case group vs. 110.1 μg/L for the control group, P < 0.05), Mo (59.6 μg/L for the case group vs. 261.7 μg/L for the control group, P < 0.05), and Sn (4.5 μg/L for the case group vs. 27.3 μg/L for the control group, P < 0.05) while significantly increased Cd (25.0 μg/L for the case group vs. 15.5 μg/L for the control group, P < 0.05). The accuracy of the discriminant function established by the Fisher analysis was 91.4%. Conclusions: Patients with RCC exhibit differences in such urinary trace elements as Mg, V, Mo, Sn, and Cd with healthy populations, and the discriminant accuracy is high.

Conformational stabilization of FOX–DNA complex architecture to sensitize prostate cancer chemotherapy

The forkhead box (FOX) transcription factor is a family of tumor suppressors that negatively regulates the tumorigenesis activity of prostate cancer; stabilization of FOX–DNA complex architecture has been recognized as a new and promising strategy for sensitizing cancer chemotherapy. Here, we described a systematic method that combined in silico analysis and in vitro assay to investigate the intermolecular interaction between FOX DNA-binding domain (DBD) and its cognate DNA partner. The structural and energetic information harvested from the molecular investigation were used to guide high-throughput virtual screening against a structurally diverse, nonredundant library of natural product compounds, aiming at discovery of novel small-molecule medicines that can conformationally stabilize and promote FOX–DNA recognition and interaction. The screening identified a number of theoretically promising hits, which were then examined by using fluorescence anisotropy assay to determine their binding potency for FOX DBD domain. The antitumor activity of identified high-affinity compounds was also tested at cellular level. Structural dynamics analysis found that the small-molecule stabilizers can shift the conformational equilibrium of FOX DBD to DNA-bound state, thus promoting the protein domain to bind tightly with its DNA partner.