@article{Zhang2019,

title = {Rapid generation of chemical combinations on a magnetic digital microfluidic array},

author = {Y Zhang and T H Wang

},

url = {https://pubs.rsc.org/en/content/articlelanding/2019/ra/c9ra03469b#!divAbstract},

year  = {2019},

date = {2019-07-12},

journal = {RSC Advances},

volume = {9},

number = {38},

pages = {21741-21747},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6737,

title = {Customizing droplet contents and dynamic ranges via integrated programmable picodroplet assembler},

author = {P Zhang and A Kaushik and K Hsieh and T H Wang},

url = {https://doi.org/10.1038/s41378-019-0062-5},

doi = {10.1038/s41378-019-0062-5},

issn = {2055-7434},

year  = {2019},

date = {2019-07-01},

journal = {Microsystems & Nanoengineering},

volume = {5},

number = {1},

pages = {22},

abstract = {Droplet microfluidic technology is becoming increasingly useful for high-throughput and high-sensitivity detection of biological and biochemical reactions. Most current droplet devices function by passively discretizing a single sample subject to a homogeneous or random reagent/reaction condition into tens of thousands of picoliter-volume droplets for analysis. Despite their apparent advantages in speed and throughput, these droplet devices inherently lack the capability to customize the contents of droplets in order to test a single sample against multiple reagent conditions or multiple samples against multiple reagents. In order to incorporate such combinatorial capability into droplet platforms, we have developed the fully Integrated Programmable Picodroplet Assembler. Our platform is capable of generating customized picoliter-volume droplet groups from nanoliter-volume plugs which are assembled in situ on demand. By employing a combination of microvalves and flow-focusing-based discretization, our platform can be used to precisely control the content and volume of generated nanoliter-volume plugs, and thereafter the content and the effective dynamic range of picoliter-volume droplets. Furthermore, we can use a single integrated device for continuously generating, incubating, and detecting multiple distinct droplet groups. The device successfully marries the precise control and on-demand capability of microvalve-based platforms with the sensitivity and throughput of picoliter droplet platforms in a fully automated monolithic device. The device ultimately will find important applications in single-cell and single-molecule analyses.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6709,

title = {Widespread gene transfer to malignant gliomas with In vitro-to-In vivo correlation},

author = { K. Negron and N. Khalasawi and B. Lu and C. Y. Ho and J. Lee and S. Shenoy and H. Q. Mao and T. H. Wang and J. Hanes and J. S. Suk},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30978431},

doi = {10.1016/j.jconrel.2019.04.010},

issn = {1873-4995 (Electronic) 

0168-3659 (Linking)},

year  = {2019},

date = {2019-04-09},

journal = {J Control Release},

abstract = {Gene therapy of malignant gliomas has shown a lack of clinical success to date due in part to inability of conventional gene vectors to achieve widespread gene transfer throughout highly disseminated tumor areas within the brain. Here, we demonstrate that newly engineered polymer-based DNA-loaded nanoparticles (DNA-NP) possessing small particle diameters (~50nm) and non-adhesive surface polyethylene glycol (PEG) coatings efficiently penetrate brain tumor tissue as well as healthy brain parenchyma. Specifically, this brain-penetrating nanoparticle (BPN), following intracranial administration via convection enhanced delivery (CED), provides widespread transgene expression in heathy rodent striatum and an aggressive brain tumor tissue established orthotopically in rats. The ability of BPN to efficiently traverse both tissues is of great importance as the highly invasive glioma cells infiltrated into normal brain tissue are responsible for tumor recurrence. Of note, the transgene expression within the orthotopic tumor tissue occurred preferentially in glioma cells over microglial cells. We also show that three-dimensional (3D) multicellular spheroids established with malignant glioma cells, unlike conventional two-dimensional (2D) cell cultures, serve as an excellent in vitro model reliably predicting gene vector behaviors in vivo. Briefly, DNA-NP possessing greater surface PEG coverage exhibited more uniform and higher-level transgene expression both in the 3D model and in vivo, whereas the trend was opposite in 2D culture. The finding here alerts that gene transfer studies based primarily on 2D cultures should be interpreted with caution and underscores the relevance of 3D models for screening newly engineered gene vectors prior to their in vivo evaluation.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6710,

title = {Promoter methylation of ADAMTS1 and BNC1 as potential biomarkers for early detection of pancreatic cancer in blood},

author = { M. A. L. Eissa and L. Lerner and E. Abdelfatah and N. Shankar and J. K. Canner and N. M. Hasan and V. Yaghoobi and B. Huang and Z. Kerner and F. Takaesu and C. Wolfgang and R. Kwak and M. Ruiz and M. Tam and T. R. 2nd Pisanic and C. A. Iacobuzio-Donahue and R. H. Hruban and J. He and T. H. Wang and L. D. Wood and A. Sharma and N. Ahuja},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30953539},

doi = {10.1186/s13148-019-0650-0},

issn = {1868-7083 (Electronic) 

1868-7075 (Linking)},

year  = {2019},

date = {2019-04-05},

journal = {Clin Epigenetics},

volume = {11},

number = {1},

pages = {59},

abstract = {BACKGROUND: Despite improvements in cancer management, most pancreatic cancers are still diagnosed at an advanced stage. We have recently identified promoter DNA methylation of the genes ADAMTS1 and BNC1 as potential blood biomarkers of pancreas cancer. In this study, we validate this biomarker panel in peripheral cell-free tumor DNA of patients with pancreatic cancer. RESULTS: Sensitivity and specificity for each gene are as follows: ADAMTS1 87.2% and 95.8% (AUC = 0.91; 95% CI 0.71-0.86) and BNC1 64.1% and 93.7% (AUC = 0.79; 95% CI 0.63-0.78). When using methylation of either gene as a combination panel, sensitivity increases to 97.3% and specificity to 91.6% (AUC = 0.95; 95% CI 0.77-0.90). Adding pre-operative CA 19-9 values to the combined two-gene methylation panel did not improve sensitivity. Methylation of ADAMTS1 was found to be positive in 87.5% (7/8) of stage I, 77.8% (7/9) of stage IIA, and 90% (18/20) of stage IIB disease. Similarly, BNC1 was positive in 62.5% (5/8) of stage I patients, 55.6% (5/9) of stage IIA, and 65% (13/20) of patients with stage IIB disease. The two-gene panel (ADAMTS1 and/or BNC1) was positive in 100% (8/8) of stage I, 88.9% (8/9) of stage IIA, and 100% (20/20) of stage IIB disease. The sensitivity and specificity of the two-gene panel for localized pancreatic cancer (stages I and II), where the cancer is eligible for surgical resection with curative potential, was 94.8% and 91.6% respectively. Additionally, the two-gene panel exhibited an AUC of 0.95 (95% CI 0.90-0.98) compared to 57.1% for CA 19-9 alone. CONCLUSION: The methylation status of ADAMTS1 and BNC1 in cfDNA shows promise for detecting pancreatic cancer during the early stages when curative resection of the tumor is still possible. This minimally invasive blood-based biomarker panel could be used as a promising tool for diagnosis and screening in a select subset of high-risk populations.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6707,

title = {Emerging Analytical Techniques for Rapid Pathogen Identification and Susceptibility Testing},

author = { D. J. Shin and N. Andini and K. Hsieh and S. Yang and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30939033},

doi = {10.1146/annurev-anchem-061318-115529},

issn = {1936-1335 (Electronic) 

1936-1327 (Linking)},

year  = {2019},

date = {2019-04-02},

journal = {Annu Rev Anal Chem (Palo Alto Calif)},

abstract = {In the face of looming threats from multi-drug resistant microorganisms, there is a growing need for technologies that will enable rapid identification and drug susceptibility profiling of these pathogens in health care settings. In particular, recent progress in microfluidics and nucleic acid amplification is pushing the boundaries of timescale for diagnosing bacterial infections. With a diverse range of techniques and parallel developments in the field of analytical chemistry, an integrative perspective is needed to understand the significance of these developments. This review examines the scope of new developments in assay technologies grouped by key enabling domains of research. First, we examine recent development in nucleic acid amplification assays for rapid identification and drug susceptibility testing in bacterial infections. Next, we examine advances in microfluidics that facilitate acceleration of diagnostic assays via integration and scale. Lastly, recent developments in biosensor technologies are reviewed. We conclude this review with perspectives on the use of emerging concepts to develop paradigm-changing assays. Expected final online publication date for the Annual Review of Analytical Chemistry Volume 12 is June 12, 2019. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6711,

title = {Methylation Biomarker Panel Performance in EsophaCap Cytology Samples for Diagnosing Barrett's Esophagus: A Prospective Validation Study},

author = { Z. Wang and S. Kambhampati and Y. Cheng and K. Ma and C. Simsek and A. H. Tieu and J. M. Abraham and X. Liu and V. Prasath and M. Duncan and A. Stark and A. Trick and H. L. Tsai and H. Wang and Y. He and M. A. Khashab and S. Ngamruengphong and E. J. Shin and T. H. Wang and S. J. Meltzer},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30670490},

doi = {10.1158/1078-0432.CCR-18-3696},

issn = {1078-0432 (Print) 

1078-0432 (Linking)},

year  = {2019},

date = {2019-04-01},

journal = {Clin Cancer Res},

volume = {25},

number = {7},

pages = {2127-2135},

abstract = {PURPOSE: Barrett's esophagus is the only known precursor of esophageal adenocarcinoma (EAC). Although endoscopy and biopsy are standard methods for Barrett's esophagus diagnosis, their high cost and risk limit their use as a screening modality. Here, we sought to develop a Barrett's esophagus detection method based on methylation status in cytology samples captured by EsophaCap using a streamlined sensitive technique, methylation on beads (MOB). EXPERIMENTAL DESIGN: We conducted a prospective cohort study on 80 patients (52 in the training set; 28 in the test set). We used MOB to extract and bisulfite-convert DNA, followed by quantitative methylation-specific PCR to assess methylation levels of 8 previously selected candidate markers. Lasso regression was applied to establish a prediction model in the training set, which was then tested on the independent test set. RESULTS: In the training set, five of eight candidate methylation biomarkers (p16, HPP1, NELL1, TAC1, and AKAP12) were significantly higher in Barrett's esophagus patients than in controls. We built a four-biomarker-plus-age lasso regression model for Barrett's esophagus diagnosis. The AUC was 0.894, with sensitivity 94.4% [95% confidence interval (CI), 71%-99%] and specificity 62.2% (95% CI, 44.6%-77.3%) in the training set. This model also performed with high accuracy for Barrett's esophagus diagnosis in an independent test set: AUC = 0.929 (P < 0.001; 95% CI, 0.810%-1%), with sensitivity=78.6% (95% CI, 48.8%-94.3%) and specificity = 92.8% (95% CI, 64.1%-99.6%). CONCLUSIONS: EsophaCap, in combination with an epigenetic biomarker panel and the MOB method, is a promising, well-tolerated, low-cost esophageal sampling strategy for Barrett's esophagus diagnosis. This approach merits further prospective studies in larger populations.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6712,

title = {Long Interspersed Nuclear Element 1 Retrotransposons Become Deregulated during the Development of Ovarian Cancer Precursor Lesions},

author = {T.R. Pisanic and Y. Wang and H. Sun and M. Considine and L. Li, T.H. Wang and T.L. Wang and I.M. Shih},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30553834},

doi = {10.1016/j.ajpath.2018.11.005},

issn = {1525-2191 (Electronic) 0002-9440 (Linking)},

year  = {2019},

date = {2019-03-01},

journal = {Am J Pathol},

volume = {189},

number = {3},

pages = {513-520},

abstract = {There is growing evidence that most high-grade serous ovarian carcinomas likely arise from local dissemination of precursor lesions of the fallopian tube. Evolution of these lesions from early p53 signatures to latter-stage, serous tubal intraepithelial carcinomas (STICs) is characterized by cytologic atypia, accumulation of somatic mutations, and genomic instability, the etiologies of which remain unclear. Long interspersed element 1 (LINE-1) retrotransposon is expressed in many carcinomas, including high-grade serous ovarian carcinoma, where it contributes to genomic instability; however, the timing of LINE-1 activation during this evolution has yet to be elucidated. In this study, we assessed LINE-1 open reading frame 1 protein expression in 12 p53 signature lesions, 32 STICs, and 112 various types of ovarian cancers via immunohistochemical staining and examined LINE-1 promoter methylation in representative cases. We found that 78% and 57% of STICs, with and without concurrent ovarian carcinomas, respectively, exhibited intense LINE-1 immunoreactivity compared with adjacent, normal-appearing fallopian tube epithelium. Hypomethylation of the LINE-1 promoter was found in all STICs exhibiting overexpression. None of the 12 p53 signatures demonstrated significant LINE-1 expression. In ovarian cancer, 84 (75%) of 112 ovarian carcinomas overexpressed LINE-1. Our results indicate that LINE-1 retrotransposons often become deregulated during progression of ovarian cancer precursor lesions from the p53 signature to STIC stages and remain highly expressed in carcinoma.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6708,

title = {Nanotube assisted microwave electroporation for single cell pathogen identification and antimicrobial susceptibility testing},

author = { J. Gao and H. Li and P. Torab and K. E. Mach and D. W. Craft and N. J. Thomas and C. M. Puleo and J. C. Liao and T. H. Wang and P. K. Wong},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30794964},

doi = {10.1016/j.nano.2019.01.015},

issn = {1549-9642 (Electronic) 

1549-9634 (Linking)},

year  = {2019},

date = {2019-02-20},

journal = {Nanomedicine},

volume = {17},

pages = {246-253},

abstract = {A nanotube assisted microwave electroporation (NAME) technique is demonstrated for delivering molecular biosensors into viable bacteria for multiplex single cell pathogen identification to advance rapid diagnostics in clinical microbiology. Due to the small volume of a bacterial cell (~femtoliter), the intracellular concentration of the target molecule is high, which results in a strong signal for single cell detection without amplification. The NAME procedure can be completed in as little as 30 minutes and can achieve over 90% transformation efficiency. We demonstrate the feasibility of NAME for identifying clinical isolates of bloodborne and uropathogenic pathogens and detecting bacterial pathogens directly from patient's samples. In conjunction with a microfluidic single cell trapping technique, NAME allows single cell pathogen identification and antimicrobial susceptibility testing concurrently. Using this approach, the time for microbiological analysis reduces from days to hours, which will have a significant impact on the clinical management of bacterial infections.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6704,

title = {Versatile Analysis of DNA-Biomolecule Interactions in Solution by Hydrodynamic Separation and Single Molecule Detection},

author = { S. M. Friedrich and R. Bang and A. Li and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30668901},

doi = {10.1021/acs.analchem.8b04733},

issn = {1520-6882 (Electronic) 

0003-2700 (Linking)},

year  = {2019},

date = {2019-02-19},

journal = {Anal Chem},

volume = {91},

number = {4},

pages = {2822-2830},

abstract = {DNA can interact with a wide array of molecules with a range of binding affinities, stoichiometry, and size-scales. We present a sensitive, quantitative, and versatile platform for sensing and evaluating these diverse DNA-biomolecule interactions and DNA conformational changes in free solution. Single molecule free solution hydrodynamic separation utilizes differences in hydrodynamic mobility to separate bound DNA-biomolecule complexes from unbound DNA and determine the associated size change that results from binding. Single molecule detection enables highly quantitative analysis of the fraction of DNA in the bound and unbound state to characterize binding behavior including affinity, stoichiometry, and cooperativity. A stacked injection scheme increases throughput to enable practical analysis of DNA-biomolecule interactions using only picoliters of sample per measurement. To demonstrate analysis of DNA-protein interactions on a local scale, we investigate binding of the E. coli single stranded binding protein to two DNA oligos both individually and in direct competition. We show that stoichiometry and cooperativity is a function of DNA length and verify these differences in binding characteristics through direct competition. To demonstrate analysis of DNA-small molecule interactions and global conformational changes, we also assess DNA condensation with the polyamine spermidine. We use hydrodynamic mobility to evaluate the size of spermidine-condensed DNA and single molecule burst analysis to evaluate DNA packing within the condensed globules relative to free-coiled DNA. This platform thus presents a versatile tool capable of quantitative and sensitive evaluation of diverse biomolecular interactions, complex properties, and binding characteristics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6703,

title = {Multilayer microfluidic array for highly efficient sample loading and digital melt analysis of DNA methylation},

author = { C. M. O'Keefe and D. Giammanco and S. Li and T. R. Pisanic and T. J. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30623957},

doi = {10.1039/c8lc01189c},

issn = {1473-0189 (Electronic) 

1473-0189 (Linking)},

year  = {2019},

date = {2019-01-29},

journal = {Lab Chip},

volume = {19},

number = {3},

pages = {444-451},

abstract = {Liquid biopsies contain a treasure of genetic and epigenetic biomarkers that contain information for the detection and monitoring of human disease. DNA methylation is an epigenetic modification that is critical to determining cellular phenotype and often becomes altered in many disease states. In cancer, aberrant DNA methylation contributes to carcinogenesis and can profoundly affect tumor evolution, metastatic potential, and resistance to therapeutic intervention. However, current technologies are not well-suited for quantitative assessment of DNA methylation heterogeneity, especially in challenging samples such as liquid biopsies with low DNA input and high background. We present a multilayer microfluidic device for quantitative analysis of DNA methylation by digital PCR and high resolution melt (HRM). The multilayer design facilitates high-density array digitization aimed at maximizing sample loading efficiency. The platform achieves highly parallelized digital PCR-HRM-based discrimination of rare heterogeneous DNA methylation as low as 0.0001% methylated/unmethylated molecules of a classic tumor suppressor gene, CDKN2A (p14ARF).},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6705,

title = {Optimizing peptide nucleic acid probes for hybridization-based detection and identification of bacterial pathogens},

author = { K. E. Mach and A. M. Kaushik and K. Hsieh and P. K. Wong and T. H. Wang and J. C. Liao},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30656297},

doi = {10.1039/c8an02194e},

issn = {1364-5528 (Electronic) 

0003-2654 (Linking)},

year  = {2019},

date = {2019-01-18},

journal = {Analyst},

abstract = {Point-of-care (POC) diagnostics for infectious diseases have the potential to improve patient care and antibiotic stewardship. Nucleic acid hybridization is at the core of many amplification-free molecular diagnostics and detection probe configuration is key to diagnostic performance. Modified nucleic acids such as peptide nucleic acid (PNA) offer advantages compared to conventional DNA probes allowing for faster hybridization, better stability and minimal sample preparation for direct detection of pathogens. Probes with tethered fluorophore and quencher allow for solution-based assays and eliminate the need for washing steps thereby facilitating integration into microfluidic devices. Here, we compared the sensitivity and specificity of double stranded PNA probes (dsPNA) and PNA molecular beacons targeting E. coli and P. aeruginosa for direct detection of bacterial pathogens. In bulk fluid assays, the dsPNAs had an overall higher fluorescent signal and better sensitivity and specificity than the PNA beacons for pathogen detection. We further designed and tested an expanded panel of dsPNA probes for detection of a wide variety of pathogenic bacteria including probes for universal detection of eubacteria, Enterobacteriaceae family, and P. mirablis. To confirm that the advantage translated to other assay types we compared the PNA beacon and dsPNA in a prototype droplet microfluidic device. Beyond the bulk fluid assay and droplet devices, use of dsPNA probes may be advantageous in a wide variety of assays that employ homogenous nucleic acid hybridization.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6646,

title = {Methylomic Analysis of Ovarian Cancers Identifies Tumor-Specific Alterations Readily Detectable in Early Precursor Lesions},

author = { T. R. 2nd Pisanic and L. M. Cope and S. F. Lin and T. T. Yen and P. Athamanolap and R. Asaka and K. Nakayama and A. N. Fader and T. H. Wang and I. M. Shih and T. L. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30108103},

doi = {10.1158/1078-0432.CCR-18-1199},

issn = {1078-0432 (Print) 

1078-0432 (Linking)},

year  = {2018},

date = {2018-12-15},

journal = {Clin Cancer Res},

volume = {24},

number = {24},

pages = {6536-6547},

abstract = {PURPOSE: High-grade serous ovarian carcinoma (HGSOC) typically remains undiagnosed until advanced stages when peritoneal dissemination has already occurred. Here, we sought to identify HGSOC-specific alterations in DNA methylation and assess their potential to provide sensitive and specific detection of HGSOC at its earliest stages. EXPERIMENTAL DESIGN: MethylationEPIC genome-wide methylation analysis was performed on a discovery cohort comprising 23 HGSOC, 37 non-HGSOC malignant, and 36 histologically unremarkable gynecologic tissue samples. The resulting data were processed using selective bioinformatic criteria to identify regions of high-confidence HGSOC-specific differential methylation. Quantitative methylation-specific real-time PCR (qMSP) assays were then developed for 8 of the top-performing regions and analytically validated in a cohort of 90 tissue samples. Lastly, qMSP assays were used to assess and compare methylation in 30 laser-capture microdissected (LCM) fallopian tube epithelia samples obtained from cancer-free and serous tubal intraepithelial carcinoma (STIC) positive women. RESULTS: Bioinformatic selection identified 91 regions of robust, HGSOC-specific hypermethylation, 23 of which exhibited an area under the receiver-operator curve (AUC) value >/= 0.9 in the discovery cohort. Seven of 8 top-performing regions demonstrated AUC values between 0.838 and 0.968 when analytically validated by qMSP in a 90-patient cohort. A panel of the 3 top-performing genes (c17orf64, IRX2, and TUBB6) was able to perfectly discriminate HGSOC (AUC 1.0). Hypermethylation within these loci was found exclusively in LCM fallopian tube epithelia from women with STIC lesions, but not in cancer-free fallopian tubes. CONCLUSIONS: A panel of methylation biomarkers can be used to accurately identify HGSOC, even at precursor stages of the disease.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6627,

title = {Fluorescence spectroscopic detection and measurement of single telomere molecules},

author = { C. W. Beh and Y. Zhang and Y. L. Zheng and B. Sun and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30010842},

doi = {10.1093/nar/gky627},

issn = {1362-4962 (Electronic) 

0305-1048 (Linking)},

year  = {2018},

date = {2018-11-02},

journal = {Nucleic Acids Res},

volume = {46},

number = {19},

pages = {e117},

abstract = {Telomeres are the end-caps of chromosomes that serve to protect the integrity of the genome. Below certain critical lengths, the telomeres can no longer fulfill their protective function, and chromosomal instability ensues. Telomeres shorten during normal cell division due to the end replication problem and are implicated in the development of various aging-associated diseases, including cancer. Telomere length has the potential to serve as a useful biomarker in the field of aging and cancer. However, existing methods of telomere measurement are either too laborious, unable to provide absolute measurement of individual telomere lengths, or limited to certain chromosomes or cell types. Here, we describe an easy single-molecule, fluorescence spectroscopic method for measuring the length of telomeres that permits the profiling of absolute telomere lengths in any DNA sample. We have demonstrated the accurate detection of telomeres as short as 100 bp using cloned telomere standards, and have profiled telomere lengths in human cancer cell lines and primary cells. Since this method allows direct comparison between samples, it could greatly improve the clinical utility of telomere biomarkers.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6502,

title = {Droplet microfluidics for high-sensitivity and high-throughput detection and screening of disease biomarkers},

author = { A. M. Kaushik and K. Hsieh and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/29797414},

doi = {10.1002/wnan.1522},

issn = {1939-0041 (Electronic) 

1939-0041 (Linking)},

year  = {2018},

date = {2018-11-01},

journal = {Wiley Interdiscip Rev Nanomed Nanobiotechnol},

volume = {10},

number = {6},

pages = {e1522},

abstract = {Biomarkers are nucleic acids, proteins, single cells, or small molecules in human tissues or biological fluids whose reliable detection can be used to confirm or predict disease and disease states. Sensitive detection of biomarkers is therefore critical in a variety of applications including disease diagnostics, therapeutics, and drug screening. Unfortunately for many diseases, low abundance of biomarkers in human samples and low sample volumes render standard benchtop platforms like 96-well plates ineffective for reliable detection and screening. Discretization of bulk samples into a large number of small volumes (fL-nL) via droplet microfluidic technology offers a promising solution for high-sensitivity and high-throughput detection and screening of biomarkers. Several microfluidic strategies exist for high-throughput biomarker digitization into droplets, and these strategies have been utilized by numerous droplet platforms for nucleic acid, protein, and single-cell detection and screening. While the potential of droplet-based platforms has led to burgeoning interest in droplets, seamless integration of sample preparation technologies and automation of platforms from biological sample to answer remain critical components that can render these platforms useful in the clinical setting in the near future. This article is categorized under: Diagnostic Tools > Biosensing Diagnostic Tools > Diagnostic Nanodevices Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Infectious Disease.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6736,

title = {Ratiometric Fluorescence Coding for Multiplex Nucleic Acid Amplification Testing},

author = { Y. Zhang and L. Chen and K. Hsieh and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30251853},

doi = {10.1021/acs.analchem.8b03266},

issn = {1520-6882 (Electronic) 

0003-2700 (Linking)},

year  = {2018},

date = {2018-10-16},

journal = {Anal Chem},

volume = {90},

number = {20},

pages = {12180-12186},

abstract = {Although nucleic acid amplification testing (NAAT) has become the cornerstone for molecular diagnosis of diseases, expanding the multiplexed detection capacity of NAAT remains an important objective. To this end, encoding each nucleic acid target with a specific fluorescently labeled probe has been the most mature approach for multiplexed detection. Unfortunately, the number of targets that can be differentiated via this one-target-one-fluorophore multiplexed detection approach is restricted by spectral overlaps between fluorophores. In response, we present herein a new multiplexed detection approach termed ratiometric fluorescence coding, in which we encode each nucleic acid target with a specific ratio between two standard fluorophores. In ratiometric fluorescence coding, we employ the padlock probe chemistry to encode each nucleic acid target with a specific number of binding sites for two probes labeled with different fluorophores. Coupling the padlock probes with either rolling circle amplification (RCA) or hyperbranched rolling circle amplification (HRCA), we transform each nucleic acid target into a specific template that allows hybridization with the fluorescently labeled probes at predesigned ratios, thereby achieving multiplexed detection. For demonstration, we detected DNA targets from six infectious diseases and demonstrated the potential for further expanding the multiplexing capability of our approach. With further development, ratiometric fluorescence coding has the potential to enable highly multiplexed detection of nucleic acid targets and facilitate molecular diagnosis of diseases.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6607,

title = {A "Culture" Shift: Broad Bacterial Detection, Identification, and Antimicrobial Susceptibility Testing Directly from Whole Blood},

author = { N. Andini and A. Hu and L. Zhou and S. Cogill and T. H. Wang and C. T. Wittwer and S. Yang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30087140},

doi = {10.1373/clinchem.2018.290189},

issn = {1530-8561 (Electronic) 

0009-9147 (Linking)},

year  = {2018},

date = {2018-10-01},

journal = {Clin Chem},

volume = {64},

number = {10},

pages = {1453-1462},

abstract = {BACKGROUND: The time required for bloodstream pathogen detection, identification (ID), and antimicrobial susceptibility testing (AST) does not satisfy the acute needs of disease management. Conventional methods take up to 3 days for ID and AST. Molecular diagnostics have reduced times for ID, but their promise to supplant culture is unmet because AST times remain slow. We developed a combined quantitative PCR (qPCR)-based ID+AST assay with sequential detection, ID, and AST of leading nosocomial bacterial pathogens. METHODS: ID+AST was performed on whole blood samples by (a) removing blood cells, (b) brief bacterial enrichment, (c) bacterial detection and ID, and (d) species-specific antimicrobial treatment. Broad-spectrum qPCR of the internal transcribed spacer between the 16S and 23S was amplified for detection. High-resolution melting identified the species with a curve classifier. AST was enabled by Ct differences between treated and untreated samples. RESULTS: A detection limit of 1 CFU/mL was achieved for Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. All species were accurately identified by unique melting curves. Antimicrobial minimum inhibitory concentrations were identified with Ct differences of >/=1 cycle. Using an RNA target allowed reduction of AST incubation time from 60 min to 5 min. Rapid-cycle amplification reduced qPCR times by 83% to 30 min. CONCLUSIONS: Combined, sequential ID+AST protocols allow rapid and reliable detection, ID, and AST for the diagnosis of bloodstream infections, enabling conversion of empiric to targeted therapy by the second dose of antimicrobials.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6641,

title = {Direct-qPCR Assay for Coupled Identification and Antimicrobial Susceptibility Testing of Neisseria gonorrhoeae},

author = { L. Chen and D. J. Shin and S. Zheng and J. H. Melendez and C. A. Gaydos and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/29999304},

doi = {10.1021/acsinfecdis.8b00104},

issn = {2373-8227 (Electronic) 

2373-8227 (Linking)},

year  = {2018},

date = {2018-09-14},

journal = {ACS Infect Dis},

volume = {4},

number = {9},

pages = {1377-1384},

abstract = {Multidrug-resistant gonorrhea has become an urgent issue for global public health. As the causative agent of gonorrhea, Neisseria gonorrhoeae, has been progressively developing resistance to nearly all prescribed antimicrobial drugs, monitoring its antimicrobial resistance on a broader scale has become a crucial agenda for effective antibiotic stewardship. Unfortunately, gold standard antimicrobial susceptibility testing (AST) relies on time and labor-intensive phenotypic assays, which lag behind the current diagnostic workflow for N. gonorrhoeae identification based on nucleic acid amplification tests (NAAT). Newer assay technologies based on NAAT can rapidly identify N. gonorrhoeae from clinical specimen but fundamentally lack the capacity to provide phenotypic AST information. Herein, we propose a direct-quantitative PCR (direct-qPCR) assay that enables pathogen-specific identification and phenotypic AST via quantitative measurement of N. gonorrhoeae growth directly from a liquid medium without any sample preprocessing. The assay has an analytical sensitivity of 10(2) CFU/mL and is highly specific to N. gonorrhoeae in the presence of urogenital flora and clinical swab eluent. We tested seven N. gonorrhoeae strains against three antibiotic agents, penicillin, tetracycline, and ciprofloxacin, and achieved 95.2% category agreement and 85.7% essential agreement with the FDA-approved E-test. The assay presented in this work has the unique ability to identify N. gonorrhoeae and provide phenotypic AST directly from the liquid medium with cell densities as low as 10(2) CFU/mL, demonstrating an accelerated, sensitive, and scalable workflow for performing both identification and AST of N. gonorrhoeae.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6702,

title = {Facile profiling of molecular heterogeneity by microfluidic digital melt},

author = { C. M. O'Keefe and T. R. 2nd Pisanic and H. Zec and M. J. Overman and J. G. Herman and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30263958},

doi = {10.1126/sciadv.aat6459},

issn = {2375-2548 (Electronic) 

2375-2548 (Linking)},

year  = {2018},

date = {2018-09-01},

journal = {Sci Adv},

volume = {4},

number = {9},

pages = {eaat6459},

abstract = {This work presents a digital microfluidic platform called HYPER-Melt (high-density profiling and enumeration by melt) for highly parallelized copy-by-copy DNA molecular profiling. HYPER-Melt provides a facile means of detecting and assessing sequence variations of thousands of individual DNA molecules through digitization in a nanowell microchip array, allowing amplification and interrogation of individual template molecules by detecting HRM fluorescence changes due to sequence-dependent denaturation. As a model application, HYPER-Melt is used here for the detection and assessment of intermolecular heterogeneity of DNA methylation within the promoters of classical tumor suppressor genes. The capabilities of this platform are validated through serial dilutions of mixed epialleles, with demonstrated detection limits as low as 1 methylated variant in 2 million unmethylated templates (0.00005%) of a classic tumor suppressor gene, CDKN2A (p14(ARF)). The clinical potential of the platform is demonstrated using a digital assay for NDRG4, a tumor suppressor gene that is commonly methylated in colorectal cancer, in liquid biopsies of healthy and colorectal cancer patients. Overall, the platform provides the depth of information, simplicity of use, and single-molecule sensitivity necessary for rapid assessment of intermolecular variation contributing to genetic and epigenetic heterogeneity for challenging applications in embryogenesis, carcinogenesis, and rare biomarker detection.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6643,

title = {Simple and Precise Counting of Viable Bacteria by Resazurin-Amplified Picoarray Detection},

author = { K. Hsieh and H. C. Zec and L. Chen and A. M. Kaushik and K. E. Mach and J. C. Liao and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/29969556},

doi = {10.1021/acs.analchem.8b02096},

issn = {1520-6882 (Electronic) 

0003-2700 (Linking)},

year  = {2018},

date = {2018-08-07},

journal = {Anal Chem},

volume = {90},

number = {15},

pages = {9449-9456},

abstract = {Simple, fast, and precise counting of viable bacteria is fundamental to a variety of microbiological applications such as food quality monitoring and clinical diagnosis. To this end, agar plating, microscopy, and emerging microfluidic devices for single bacteria detection have provided useful means for counting viable bacteria, but they also have their limitations ranging from complexity, time, and inaccuracy. We present herein our new method RAPiD (Resazurin-Amplified Picoarray Detection) for addressing this important problem. In RAPiD, we employ vacuum-assisted sample loading and oil-driven sample digitization to stochastically confine single bacteria in Picoarray, a microfluidic device with picoliter-sized isolation chambers (picochambers), in <30 s with only a few minutes of hands-on time. We add AlamarBlue, a resazurin-based fluorescent dye for bacterial growth, in our assay to accelerate the detection of "microcolonies" proliferated from single bacteria within picochambers. Detecting fluorescence in picochambers as an amplified surrogate for bacterial cells allows us to count hundreds of microcolonies with a single image taken via wide-field fluorescence microscopy. We have also expanded our method to practically test multiple titrations from a single bacterial sample in parallel. Using this expanded "multi-RAPiD" strategy, we can quantify viable cells in E. coli and S. aureus samples with precision in approximately 3 h, illustrating RAPiD as a promising new method for counting viable bacteria for microbiological applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN6700,

title = {Rapid Microbiology Screening in Pharmaceutical Workflows},

author = { C. Surrette and B. Scherer and A. Corwin and G. Grossmann and A. M. Kaushik and K. Hsieh and P. Zhang and J. C. Liao and P. K. Wong and T. H. Wang and C. M. Puleo},

url = {https://www.ncbi.nlm.nih.gov/pubmed/30027813},

doi = {10.1177/2472630318779758},

issn = {2472-6311 (Electronic) 

2472-6303 (Linking)},

year  = {2018},

date = {2018-08-01},

journal = {SLAS Technol},

volume = {23},

number = {4},

pages = {387-394},

abstract = {Recently advances in miniaturization and automation have been utilized to rapidly decrease the time to result for microbiology testing in the clinic. These advances have been made due to the limitations of conventional culture-based microbiology methods, including agar plate and microbroth dilution, which have long turnaround times and require physicians to treat patients empirically with antibiotics before test results are available. Currently, there exist similar limitations in pharmaceutical sterility and bioburden testing, where the long turnaround times associated with standard microbiology testing drive costly inefficiencies in workflows. These include the time lag associated with sterility screening within drug production lines and the warehousing cost and time delays within supply chains during product testing. Herein, we demonstrate a proof-of-concept combination of a rapid microfluidic assay and an efficient cell filtration process that enables a path toward integrating rapid tests directly into pharmaceutical microbiological screening workflows. We demonstrate separation and detection of Escherichia coli directly captured and analyzed from a mammalian (i.e., CHO) cell culture with a 3.0 h incubation. The demonstration is performed using a membrane filtration module that is compatible with sampling from bioreactors, enabling in-line sampling and process monitoring.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3210,

title = {Quantum dots in diagnostics and detection: principles and paradigms},

author = { T. R. 2nd Pisanic and Y. Zhang and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/24770716},

doi = {10.1039/c4an00294f},

issn = {1364-5528 (Electronic) 

0003-2654 (Linking)},

year  = {2014},

date = {2014-06-21},

journal = {Analyst},

volume = {139},

number = {12},

pages = {2968-81},

abstract = {Quantum dots are semiconductor nanocrystals that exhibit exceptional optical and electrical behaviors not found in their bulk counterparts. Following seminal work in the development of water-soluble quantum dots in the late 1990's, researchers have sought to develop interesting and novel ways of exploiting the extraordinary properties of quantum dots for biomedical applications. Since that time, over 10,000 articles have been published related to the use of quantum dots in biomedicine, many of which regard their use in detection and diagnostic bioassays. This review presents a didactic overview of fundamental physical phenomena associated with quantum dots and paradigm examples of how these phenomena can and have been readily exploited for manifold uses in nanobiotechnology with a specific focus on their implementation in in vitro diagnostic assays and biodetection.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3215,

title = {Droplet Array Platform for High-Resolution Melt Analysis of DNA Methylation Density},

author = { P. Athamanolap and D. J. Shin and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/24114798},

doi = {10.1177/2211068213507923},

issn = {2211-0690 (Electronic) 

2211-0682 (Linking)},

year  = {2014},

date = {2014-06-01},

journal = {J Lab Autom},

volume = {19},

number = {3},

pages = {304-12},

abstract = {High-resolution melting (HRM) has garnered significant interest as an analytical technique for a number of applications, including DNA methylation detection, due to its inherent sensitivity and robustness. In this study, we describe a miniaturized assay platform for quantitative methylation density analysis using a microfluidic droplet array cartridge. We demonstrate that the DNA methylation level of the RASSF1A promoter can be directly analyzed using HRM. PCR products were generated by amplifying bisulfite-treated DNA with varying CpG densities using CpG island-flanking primer sets. Subsequent HRM analysis on the miniaturized droplet platform shows distinct melting curve profiles associated with methylation levels, which was verified using a conventional benchtop PCR-HRM system. The characteristic melting temperature (Tm) of the PCR products was used to directly quantify the respective levels of DNA methylation density. Our approach provides a key advantage over current gold standard methods such as methylation-specific PCR (MSP), which are incapable of providing specific information regarding the overall methylation density of the target genes. The miniaturized platform establishes a practical approach to methylation density profiling from multiple DNA samples with a potential application in point-of-care diagnostics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3211,

title = {Elimination of ligation dependent artifacts in T4 RNA ligase to achieve high efficiency and low bias microRNA capture},

author = { Y. Song and K. J. Liu and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/24722341},

doi = {10.1371/journal.pone.0094619},

issn = {1932-6203 (Electronic) 

1932-6203 (Linking)},

year  = {2014},

date = {2014-04-12},

journal = {PLoS One},

volume = {9},

number = {4},

pages = {e94619},

abstract = {Adapter ligation is a critical first step in many microRNA analysis methods including microarray, qPCR, and sequencing. Previous studies have shown that ligation bias can have dramatic effects on both the fidelity of expression profiles and reproducibility across samples. We have developed a method for high efficiency and low bias microRNA capture by 3' adapter ligation using T4 RNA ligase that does not require pooled adapters. Using a panel of 20 microRNA, we investigated the effects of ligase type, PEG concentration, ligase amount, adapter concentration, incubation time, incubation temperature, and adapter design on capture efficiency and bias. Of these factors, high PEG% was found to be critical in suppressing ligation bias. We obtained high average capture efficiency and low CV across the 20 microRNA panel, both in idealized buffer conditions (86% +/- 10%) and total RNA spiking conditions (64% +/- 17%). We demonstrate that this method is reliable across microRNA species that previous studies have had difficulty capturing and that our adapter design performs significantly better than the common adapter designs. Further, we demonstrate that the optimization methodology must be specifically designed for minimizing bias in order to obtain the ideal reaction parameters.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3213,

title = {Large enhancement of quantum dot fluorescence by highly scalable nanoporous gold},

author = { L. Zhang and Y. Song and T. Fujita and Y. Zhang and M. Chen and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/24339211},

doi = {10.1002/adma.201304503},

issn = {1521-4095 (Electronic) 

0935-9648 (Linking)},

year  = {2014},

date = {2014-02-26},

journal = {Adv Mater},

volume = {26},

number = {8},

pages = {1289-94},

abstract = {Dealloyed nanoporous gold (NPG) dramatically enhances quantum dot (QD) fluorescence by amplifying near-field excitation and increasing the radiative decay rate. Originating from plasmonic coupling, the fluorescence enhancement is highly dependent upon the nanopore size of the NPG. In contrast to other nanoengineered metallic structures, NPG exhibits fluorescence enhancement of QDs over a large substrate surface.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3212,

title = {Pressure induced lung injury in a novel in vitro model of the alveolar interface: protective effect of dexamethasone},

author = { D. D. Nalayanda and W. B. Fulton and P. M. Colombani and T. H. Wang and F. Abdullah},

url = {https://www.ncbi.nlm.nih.gov/pubmed/24439582},

doi = {10.1016/j.jpedsurg.2013.09.030},

issn = {1531-5037 (Electronic) 

0022-3468 (Linking)},

year  = {2014},

date = {2014-01-01},

journal = {J Pediatr Surg},

volume = {49},

number = {1},

pages = {61-5; discussion 65},

abstract = {PURPOSE: The lungs of infants born with congenital diaphragmatic hernia suffer from immaturity as well as the short and long term consequences of ventilator-induced lung injury, including chronic lung disease. Antenatal and postnatal steroids are among current strategies promoted to treat premature lungs and limit long term morbidity. Although studied in whole-animal models, insight into ventilator-induced injury at the alveolar-capillary interface as well as the benefits of steroids, remains limited. The present study utilizes a multi-fluidic in vitro model of the alveolar-interface to analyze membrane disruption from compressive aerodynamic forces in dexamethasone-treated cultures. METHODS: Human alveolar epithelial cell lines, H441 and A549, were cultured in a custom-built chamber under constant aerodynamic shear followed by introduction of pressure stimuli with and without dexamethasone (0.1muM). On-chip bioelectrical measurements were noted to track changes to the cellular surface and live-dead assay to ascertain cellular viability. RESULTS: Pressure-exposed alveolar cultures demonstrated a significant drop in TEER that was less prominent with an underlying extracellular-matrix coating. Addition of dexamethasone resulted in increased alveolar layer integrity demonstrated by higher TEER values. Furthermore, dexamethasone-treated cells exhibited faster recovery, and the effects of pressure appeared to be mitigated in both cell types. CONCLUSION: Using a novel in vitro model of the alveolus, we demonstrate a dose-response relationship between pressure application and loss of alveolar layer integrity. This effect appears to be alleviated by dexamethasone and matrix sub-coating.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3214,

title = {Serial dilution via surface energy trap-assisted magnetic droplet manipulation},

author = { Y. Zhang and D. J. Shin and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/24162777},

doi = {10.1039/c3lc50915j},

issn = {1473-0189 (Electronic) 

1473-0189 (Linking)},

year  = {2013},

date = {2013-12-21},

journal = {Lab Chip},

volume = {13},

number = {24},

pages = {4827-31},

abstract = {This paper demonstrates a facile method of generating precise serial dilutions in the form of droplets on an open surface platform. The method relies on the use of surface energy traps (SETs), etched areas of high surface energy on a Teflon coated glass substrate, to assist in the magnetic manipulation of droplets to meter and dispense liquid of defined volumes for the preparation of serial dilutions. The volume of the dispensed liquid can be precisely controlled by the size of the SETs, facilitating generation of concentration profiles of high linearity. We have applied this approach to the generation of serial dilutions of antibiotics for anti-microbial susceptibility testing (AST).},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3220,

title = {Topography-assisted electromagnetic platform for blood-to-PCR in a droplet},

author = { C. H. Chiou and D. J. Shin and Y. Zhang and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/23835223},

doi = {10.1016/j.bios.2013.06.011},

issn = {1873-4235 (Electronic) 

0956-5663 (Linking)},

year  = {2013},

date = {2013-12-15},

journal = {Biosens Bioelectron},

volume = {50},

pages = {91-9},

abstract = {This paper presents an electromagnetically actuated platform for automated sample preparation and detection of nucleic acids. The proposed platform integrates nucleic acid extraction using silica-coated magnetic particles with real-time polymerase chain reaction (PCR) on a single cartridge. Extraction of genomic material was automated by manipulating magnetic particles in droplets using a series of planar coil electromagnets assisted by topographical features, enabling efficient fluidic processing over a variety of buffers and reagents. The functionality of the platform was demonstrated by performing nucleic acid extraction from whole blood, followed by real-time PCR detection of KRAS oncogene. Automated sample processing from whole blood to PCR-ready droplet was performed in 15 min. We took a modular approach of decoupling the modules of magnetic manipulation and optical detection from the device itself, enabling a low-complexity cartridge that operates in tandem with simple external instruments.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3216,

title = {Novel methylation biomarker panel for the early detection of pancreatic cancer},

author = { J. M. Yi and A. A. Guzzetta and V. J. Bailey and S. R. Downing and L. Van Neste and K. B. Chiappinelli and B. P. Keeley and A. Stark and A. Herrera and C. Wolfgang and E. P. Pappou and C. A. Iacobuzio-Donahue and M. G. Goggins and J. G. Herman and T. H. Wang and S. B. Baylin and N. Ahuja},

url = {https://www.ncbi.nlm.nih.gov/pubmed/24088737},

doi = {10.1158/1078-0432.CCR-12-3224},

issn = {1078-0432 (Print) 

1078-0432 (Linking)},

year  = {2013},

date = {2013-12-01},

journal = {Clin Cancer Res},

volume = {19},

number = {23},

pages = {6544-6555},

abstract = {PURPOSE: Pancreatic cancer is the fourth leading cause of cancer deaths and there currently is no reliable modality for the early detection of this disease. Here, we identify cancer-specific promoter DNA methylation of BNC1 and ADAMTS1 as a promising biomarker detection strategy meriting investigation in pancreatic cancer. EXPERIMENTAL DESIGN: We used a genome-wide pharmacologic transcriptome approach to identify novel cancer-specific DNA methylation alterations in pancreatic cancer cell lines. Of eight promising genes, we focused our studies on BNC1 and ADAMTS1 for further downstream analysis, including methylation and expression. We used a nanoparticle-enabled methylation on beads (MOB) technology to detect early-stage pancreatic cancers by analyzing DNA methylation in patient serum. RESULTS: We identified two novel genes, BNC1 (92%) and ADAMTS1 (68%), that showed a high frequency of methylation in pancreatic cancers (n = 143), up to 100% in PanIN-3 and 97% in stage I invasive cancers. Using the nanoparticle-enabled MOB technology, these alterations could be detected in serum samples (n = 42) from patients with pancreatic cancer, with a sensitivity for BNC1 of 79% [95% confidence interval (CI), 66%-91%] and for ADAMTS1 of 48% (95% CI, 33%-63%), whereas specificity was 89% for BNC1 (95% CI, 76%-100%) and 92% for ADAMTS1 (95% CI, 82%-100%). Overall sensitivity using both markers is 81% (95% CI, 69%-93%) and specificity is 85% (95% CI, 71%-99%). CONCLUSIONS: Promoter DNA methylation of BNC1 and ADAMTS1 is a potential biomarker to detect early-stage pancreatic cancers. Assaying the promoter methylation status of these genes in circulating DNA from serum is a promising strategy for early detection of pancreatic cancer and has the potential to improve mortality from this disease.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3218,

title = {Extraction and processing of circulating DNA from large sample volumes using methylation on beads for the detection of rare epigenetic events},

author = { B. Keeley and A. Stark and T. R. 2nd Pisanic and R. Kwak and Y. Zhang and J. Wrangle and S. Baylin and J. Herman and N. Ahuja and M. V. Brock and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/23911908},

doi = {10.1016/j.cca.2013.07.023},

issn = {1873-3492 (Electronic) 

0009-8981 (Linking)},

year  = {2013},

date = {2013-10-21},

journal = {Clin Chim Acta},

volume = {425},

pages = {169-75},

abstract = {The use of methylated tumor-specific circulating DNA has shown great promise as a potential cancer biomarker. Nonetheless, the relative scarcity of tumor-specific circulating DNA presents a challenge for traditional DNA extraction and processing techniques. Here we demonstrate a single tube extraction and processing technique dubbed "methylation on beads" that allows for DNA extraction and bisulfite conversion for up to 2 ml of plasma or serum. In comparison to traditional techniques including phenol chloroform and alcohol extraction, methylation on beads yields a 1.5- to 5-fold improvement in extraction efficiency. The technique results in far less carryover of PCR inhibitors yielding analytical sensitivity improvements of over 25-fold. The combination of improved recovery and sensitivity make possible the detection of rare epigenetic events and the development of high sensitivity epigenetic diagnostic assays.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3217,

title = {Universal digital high-resolution melt: a novel approach to broad-based profiling of heterogeneous biological samples},

author = { S. I. Fraley and J. Hardick and B. J. Masek and P. Athamanolap and R. E. Rothman and C. A. Gaydos and K. C. Carroll and T. Wakefield and T. H. Wang and S. Yang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/23935121},

doi = {10.1093/nar/gkt684},

issn = {1362-4962 (Electronic) 

0305-1048 (Linking)},

year  = {2013},

date = {2013-10-01},

journal = {Nucleic Acids Res},

volume = {41},

number = {18},

pages = {e175},

abstract = {Comprehensive profiling of nucleic acids in genetically heterogeneous samples is important for clinical and basic research applications. Universal digital high-resolution melt (U-dHRM) is a new approach to broad-based PCR diagnostics and profiling technologies that can overcome issues of poor sensitivity due to contaminating nucleic acids and poor specificity due to primer or probe hybridization inaccuracies for single nucleotide variations. The U-dHRM approach uses broad-based primers or ligated adapter sequences to universally amplify all nucleic acid molecules in a heterogeneous sample, which have been partitioned, as in digital PCR. Extensive assay optimization enables direct sequence identification by algorithm-based matching of melt curve shape and Tm to a database of known sequence-specific melt curves. We show that single-molecule detection and single nucleotide sensitivity is possible. The feasibility and utility of U-dHRM is demonstrated through detection of bacteria associated with polymicrobial blood infection and microRNAs (miRNAs) associated with host response to infection. U-dHRM using broad-based 16S rRNA gene primers demonstrates universal single cell detection of bacterial pathogens, even in the presence of larger amounts of contaminating bacteria; U-dHRM using universally adapted Lethal-7 miRNAs in a heterogeneous mixture showcases the single copy sensitivity and single nucleotide specificity of this approach.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3219,

title = {A multiphase fluidic platform for studying ventilator-induced injury of the pulmonary epithelial barrier},

author = { D. D. Nalayanda and W. B. Fulton and T. H. Wang and F. Abdullah},

url = {https://www.ncbi.nlm.nih.gov/pubmed/23835753},

doi = {10.1039/c3ib40037a},

issn = {1757-9708 (Electronic) 

1757-9694 (Linking)},

year  = {2013},

date = {2013-09-01},

journal = {Integr Biol (Camb)},

volume = {5},

number = {9},

pages = {1141-8},

abstract = {Mechanical ventilation has been a critical part of basic life support for many years, with almost one-third of all patients in the intensive care unit requiring the aid. However studies over the past two decades have indicated that ventilators have the potential to cause or aggravate pulmonary injury. The lung with its anatomically complex architecture and unique amalgam of cell types and interfaces is very difficult to replicate in vitro. This study is focused on mimicking the distal unit of the alveolus in developing an analytical platform for evaluating cellular interactions and response to mechanostimulation. The prototype developed incorporates the ability to expose alveolar cells to sustained periods of supra-physiological pressure when cultured at an air-liquid interface with constant air-flow on the apical side and medium replenishment on basolateral surfaces. The in vitro evaluation of the alveolar, A549 and H441, cells indicated disruption of the cell layer integrity in response to sustained pressure application. The results indicate a magnitude- and duration-dependent response among both the cell types.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3222,

title = {Micro and nanotechnologies enhanced biomolecular sensing},

author = { T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/25586258},

doi = {10.3390/bios3030283},

issn = {2079-6374 (Print) 

2079-6374 (Linking)},

year  = {2013},

date = {2013-07-05},

journal = {Biosensors (Basel)},

volume = {3},

number = {3},

pages = {283-5},

abstract = {This editorial summarizes some of the recent advances of micro and nanotechnology-based tools and devices for biomolecular detection. These include the incorporation of nanomaterials into a sensor surface or directly interfacing with molecular probes to enhance target detection via more rapid and sensitive responses, and the use of self-assembled organic/inorganic nanocomposites that inhibit exceptional spectroscopic properties to enable facile homogenous assays with efficient binding kinetics. Discussions also include some insight into microfluidic principles behind the development of an integrated sample preparation and biosensor platform toward a miniaturized and fully functional system for point of care applications.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3221,

title = {Full-range magnetic manipulation of droplets via surface energy traps enables complex bioassays},

author = { Y. Zhang and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/23529938},

doi = {10.1002/adma.201300383},

issn = {1521-4095 (Electronic) 

0935-9648 (Linking)},

year  = {2013},

date = {2013-06-04},

journal = {Adv Mater},

volume = {25},

number = {21},

pages = {2903-8},

abstract = {Manipulating droplets on an open surface promises an easier, more flexible, and more scalable platform of liquid control, than does microchannel-based fluidics. In this report, a surface-energy-trap-enabled magnetic droplet handling platform is introduced that is capable of comprehensive droplet manipulations, including droplet dispensing, transport, fusion, and particle extraction.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3223,

title = {Single quantum dot analysis enables multiplexed point mutation detection by gap ligase chain reaction},

author = { Y. Song and Y. Zhang and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/23239594},

doi = {10.1002/smll.201202242},

issn = {1613-6829 (Electronic) 

1613-6810 (Linking)},

year  = {2013},

date = {2013-04-08},

journal = {Small},

volume = {9},

number = {7},

pages = {1096-105},

abstract = {Gene point mutations present important biomarkers for genetic diseases. However, existing point mutation detection methods suffer from low sensitivity, specificity, and a tedious assay processes. In this report, an assay technology is proposed which combines the outstanding specificity of gap ligase chain reaction (Gap-LCR), the high sensitivity of single-molecule coincidence detection, and the superior optical properties of quantum dots (QDs) for multiplexed detection of point mutations in genomic DNA. Mutant-specific ligation products are generated by Gap-LCR and subsequently captured by QDs to form DNA-QD nanocomplexes that are detected by single-molecule spectroscopy (SMS) through multi-color fluorescence burst coincidence analysis, allowing for multiplexed mutation detection in a separation-free format. The proposed assay is capable of detecting zeptomoles of KRAS codon 12 mutation variants with near 100% specificity. Its high sensitivity allows direct detection of KRAS mutation in crude genomic DNA without PCR pre-amplification.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3224,

title = {Discerning single molecule interactions of DNA and quantum dots},

author = { T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/23097334},

doi = {10.1002/biot.201200309},

issn = {1860-7314 (Electronic) 

1860-6768 (Linking)},

year  = {2013},

date = {2013-01-01},

journal = {Biotechnol J},

volume = {8},

number = {1},

pages = {15-6},

abstract = {Measurements of interactions between nanoparticles and DNA at the molecular level offer insight to the rationale design of nano-complexes for biomedical imaging and sensing. This commentary describes the work of Kungang Li and colleagues, who report a study of atomic force microscopy-enabled single molecule imaging of quantum dots and DNA.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3227,

title = {PDMS-glass bonding using grafted polymeric adhesive--alternative process flow for compatibility with patterned biological molecules},

author = { C. W. Beh and W. Zhou and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/22858861},

doi = {10.1039/c2lc40315c},

issn = {1473-0189 (Electronic) 

1473-0189 (Linking)},

year  = {2012},

date = {2012-10-21},

journal = {Lab Chip},

volume = {12},

number = {20},

pages = {4120-7},

abstract = {We report a novel modification of silicone elastomer polydimethylsiloxane (PDMS) with a polymer graft that allows interfacial bonding between an elastomer and glass substrate to be performed without exposure of the substrate to harsh treatment conditions, such as oxygen plasma. Organic molecules can thus be patterned within microfluidic channels and still remain functional post-bonding. In addition, after polymer grafting the PDMS can be stored in a desiccator for at least 40 days, and activated upon exposure to acidic buffer for bonding. The bonded devices remain fully bonded in excess of 80 psi driving pressure, with no signs of compromise to the bond integrity. Finally, we demonstrate the compatibility of our method with biological molecules using a proof-of-concept DNA sensing device, in which fluorescently-labelled DNA targets are successfully captured by a patterned probe in a device sealed using our method, while the pattern on a plasma-treated device was completely destroyed. Therefore, this method provides a much-needed alternative bonding process for incorporation of biological molecules in microfluidic devices.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3226,

title = {A serial sample loading system: interfacing multiwell plates with microfluidic devices},

author = { T. D. Rane and H. C. Zec and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/22885789},

doi = {10.1177/2211068212455169},

issn = {2211-0690 (Electronic) 

2211-0682 (Linking)},

year  = {2012},

date = {2012-10-01},

journal = {J Lab Autom},

volume = {17},

number = {5},

pages = {370-7},

abstract = {There is an increasing demand for novel high-throughput screening (HTS) technologies in the pharmaceutical and biotechnological industries. The robotic sample-handling techniques currently used in these industries, although fast, are still limited to operating in multiwell plates with the sample volumes per reaction in the microliter regime. Digital microfluidics offers an alternative for reduction in sample volume consumption for HTS but lacks a reliable technique for transporting a large number of samples to the microfluidic device. In this report, we develop a technique for serial delivery of sample arrays to a microfluidic device from multiwell plates, through a single sample inlet. Under this approach, a serial array of sample plugs, separated by an immiscible carrier fluid, is loaded into a capillary and delivered to a microfluidic device. Similar approaches have been attempted in the past, however, either with a slower sample loading device such as a syringe pump or vacuum-based sample loading with limited driving pressure. We demonstrated the application of our positive-pressure-based serial sample loading (SSL) system to load a series of sample plugs into a capillary. The adaptability of the SSL system to generate sample plugs with a variety of volumes in a predictable manner was also demonstrated.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3228,

title = {Droplet microfluidics for amplification-free genetic detection of single cells},

author = { T. D. Rane and H. C. Zec and C. Puleo and A. P. Lee and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/22842841},

doi = {10.1039/c2lc40537g},

issn = {1473-0189 (Electronic) 

1473-0189 (Linking)},

year  = {2012},

date = {2012-09-21},

journal = {Lab Chip},

volume = {12},

number = {18},

pages = {3341-7},

abstract = {In this article we present a novel droplet microfluidic chip enabling amplification-free detection of single pathogenic cells. The device streamlines multiple functionalities to carry out sample digitization, cell lysis, probe-target hybridization for subsequent fluorescent detection. A peptide nucleic acid fluorescence resonance energy transfer probe (PNA beacon) is used to detect 16S rRNA present in pathogenic cells. Initially the sensitivity and quantification abilities of the platform are tested using a synthetic target mimicking the actual expression level of 16S rRNA in single cells. The capability of the device to perform "sample-to-answer" pathogen detection of single cells is demonstrated using E. coli as a model pathogen.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3229,

title = {Microfluidic platform for on-demand generation of spatially indexed combinatorial droplets},

author = { H. Zec and T. D. Rane and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/22810353},

doi = {10.1039/c2lc40399d},

issn = {1473-0189 (Electronic) 

1473-0189 (Linking)},

year  = {2012},

date = {2012-09-07},

journal = {Lab Chip},

volume = {12},

number = {17},

pages = {3055-62},

abstract = {We propose a highly versatile and programmable nanolitre droplet-based platform that accepts an unlimited number of sample plugs from a multi-well plate, performs digitization of these sample plugs into smaller daughter droplets and subsequent synchronization-free, robust injection of multiple reagents into the sample daughter droplets on-demand. This platform combines excellent control of valve-based microfluidics with the high-throughput capability of droplet microfluidics. We demonstrate the functioning of a proof-of-concept device which generates combinatorial mixture droplets from a linear array of sample plugs and four different reagents, using food dyes to mimic samples and reagents. Generation of a one dimensional array of the combinatorial mixture droplets on the device leads to automatic spatial indexing of these droplets, precluding the need to include a barcode in each droplet to identify its contents. We expect this platform to further expand the range of applications of droplet microfluidics to include applications requiring a high degree of multiplexing as well as high throughput analysis of multiple samples.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3225,

title = {Quantum dot enabled molecular sensing and diagnostics},

author = { Y. Zhang and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/22916072},

doi = {10.7150/thno.4308},

issn = {1838-7640 (Electronic) 

1838-7640 (Linking)},

year  = {2012},

date = {2012-08-24},

journal = {Theranostics},

volume = {2},

number = {7},

pages = {631-54},

abstract = {Since its emergence, semiconductor nanoparticles known as quantum dots (QDs) have drawn considerable attention and have quickly extended their applicability to numerous fields within the life sciences. This is largely due to their unique optical properties such as high brightness and narrow emission band as well as other advantages over traditional organic fluorophores. New molecular sensing strategies based on QDs have been developed in pursuit of high sensitivity, high throughput, and multiplexing capabilities. For traditional biological applications, QDs have already begun to replace traditional organic fluorophores to serve as simple fluorescent reporters in immunoassays, microarrays, fluorescent imaging applications, and other assay platforms. In addition, smarter, more advanced QD probes such as quantum dot fluorescence resonance energy transfer (QD-FRET) sensors, quenching sensors, and barcoding systems are paving the way for highly-sensitive genetic and epigenetic detection of diseases, multiplexed identification of infectious pathogens, and tracking of intracellular drug and gene delivery. When combined with microfluidics and confocal fluorescence spectroscopy, the detection limit is further enhanced to single molecule level. Recently, investigations have revealed that QDs participate in series of new phenomena and exhibit interesting non-photoluminescent properties. Some of these new findings are now being incorporated into novel assays for gene copy number variation (CNV) studies and DNA methylation analysis with improved quantification resolution. Herein, we provide a comprehensive review on the latest developments of QD based molecular diagnostic platforms in which QD plays a versatile and essential role.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3230,

title = {Micro Magnetic Gyromixer for Speeding up Reactions in Droplets},

author = { Y. Zhang and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/22707926},

doi = {10.1007/s10404-011-0922-2},

issn = {1613-4982 (Print) 

1613-4982 (Linking)},

year  = {2012},

date = {2012-03-01},

journal = {Microfluid Nanofluidics},

volume = {12},

number = {5},

pages = {787-794},

abstract = {We report a novel micro magnetic gyromixer designed for accelerating mixing hence reactions in droplets. The gyromixer is fabricated with magnetite-PDMS composite using soft lithography. The mixer spins and balances itself on the droplet surface through the gyroscopic effect, rapidly homogenizing the enclosed reagents by stretching and folding internal fluid streamlines to enhance mixing. We examined the capability of the gyromixer for improving biochemical reactions in droplets by monitoring the biotin-streptavidin binding as a linker in a quantum dot fluorescence resonant energy transfer (QD-FRET) sensing system. The remotely controlled gyromixer exhibits high flexibility and potential for integration in a variety of droplet-based miniaturized total analysis systems (muTAS) to reduce turnaround times.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3231,

title = {Mapping DNA quantity into electrophoretic mobility through quantum dot nanotethers for high-resolution genetic and epigenetic analysis},

author = { Y. Zhang and K. J. Liu and T. L. Wang and M. Shih Ie and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/22136600},

doi = {10.1021/nn204377k},

issn = {1936-086X (Electronic) 

1936-0851 (Linking)},

year  = {2012},

date = {2012-01-24},

journal = {ACS Nano},

volume = {6},

number = {1},

pages = {858-64},

abstract = {Newly discovered nanoparticle properties have driven the development of novel applications and uses. We report a new observation where the electrophoretic mobility of a quantum dot/DNA nanoassembly can be precisely modulated by the degree of surface DNA conjugation. By using streptavidin-coated quantum dots (QDs) as nanotethers to gather biotin-labeled DNA into electrophoretic nanoassemblies, the QD surface charge is modulated and transformed into electrophoretic mobility shifts using standard agarose gel electrophoresis. Typical fluorescent assays quantify based on relative intensity. However, this phenomenon uses a novel approach that accurately maps DNA quantity into shifts in relative band position. This property was applied in a QD-enabled nanoassay called quantum dot electrophoretic mobility shift assay (QEMSA) that enables accurate quantification of DNA targets down to 1.1-fold (9%) changes in quantity, beyond what is achievable in qPCR. In addition to these experimental findings, an analytical model is presented to explain this behavior. Finally, QEMSA was applied to both genetic and epigenetic analysis of cancer. First, it was used to analyze copy number variation (CNV) of the RSF1/HBXAP gene, where conventional approaches for CNV analysis based on comparative genomic hybridization (CGH), microarrays, and qPCR are unable to reliably differentiate less than 2-fold changes in copy number. Then, QEMSA was used for DNA methylation analysis of the p16/CDK2A tumor suppressor gene, where its ability to detect subtle changes in methylation was shown to be superior to that of qPCR.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3233,

title = {Advances in microfluidic PCR for point-of-care infectious disease diagnostics},

author = { S. Park and Y. Zhang and S. Lin and T. H. Wang and S. Yang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/21741465},

doi = {10.1016/j.biotechadv.2011.06.017},

issn = {1873-1899 (Electronic) 

0734-9750 (Linking)},

year  = {2011},

date = {2011-11-01},

journal = {Biotechnol Adv},

volume = {29},

number = {6},

pages = {830-9},

abstract = {Global burdens from existing or emerging infectious diseases emphasize the need for point-of-care (POC) diagnostics to enhance timely recognition and intervention. Molecular approaches based on PCR methods have made significant inroads by improving detection time and accuracy but are still largely hampered by resource-intensive processing in centralized laboratories, thereby precluding their routine bedside- or field-use. Microfluidic technologies have enabled miniaturization of PCR processes onto a chip device with potential benefits including speed, cost, portability, throughput, and automation. In this review, we provide an overview of recent advances in microfluidic PCR technologies and discuss practical issues and perspectives related to implementing them into infectious disease diagnostics.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3232,

title = {Continuous dielectrophoretic bacterial separation and concentration from physiological media of high conductivity},

author = { S. Park and Y. Zhang and T. H. Wang and S. Yang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/21776517},

doi = {10.1039/c1lc20307j},

issn = {1473-0189 (Electronic) 

1473-0189 (Linking)},

year  = {2011},

date = {2011-09-07},

journal = {Lab Chip},

volume = {11},

number = {17},

pages = {2893-900},

abstract = {Biological sample processing involves purifying target analytes from various sample matrices and concentrating them to a small volume from a large volume of crude sample. This complex process is the major obstacle for developing a microfluidic diagnostic platform. In this study, we present a microfluidic device that can continuously separate and concentrate pathogenic bacterial cells from complex sample matrices such as cerebrospinal fluid and whole blood. Having overcome critical limitations of dielectrophoretic (DEP) operation in physiological media of high conductivity, we utilized target specific DEP techniques to incorporate cell separation, medium exchange, and target concentration into an integrated platform. The proposed microfluidic device can uptake mL volumes of crude biological sample and selectively concentrate target cells into a submicrolitre volume, providing ~10(4) fold of concentration. We designed the device based on the electrokinetic theory and electric field simulation, and tested the device performance with different sample types. The separation efficiency of the device was as high as 97.0% for a bead mixture in TAE buffer and 94.3% and 87.2% for E. coli in human cerebrospinal fluid and blood, respectively. A capture efficiency of 100% was achieved in the concentration chamber. With a relatively simple configuration, the proposed device provides a robust method of continuous sample processing, which can be readily integrated into a fully automated microfluidic diagnostic platform for pathogen detection and quantification.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN5137,

title = {Quantum Dots-Enabled High-Resolution Analysis of Gene Copy Number Variation},

author = { Yi Zhang and Tza-Huei Wang},

doi = {10.1109/mnano.2011.940950},

issn = {1932-4510},

year  = {2011},

date = {2011-07-04},

journal = {IEEE Nanotechnology Magazine},

volume = {5},

number = {2},

pages = {23-27},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3234,

title = {Single-molecule analysis enables free solution hydrodynamic separation using yoctomole levels of DNA},

author = { K. J. Liu and T. D. Rane and Y. Zhang and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/21504160},

doi = {10.1021/ja200279y},

issn = {1520-5126 (Electronic) 

0002-7863 (Linking)},

year  = {2011},

date = {2011-05-11},

journal = {J Am Chem Soc},

volume = {133},

number = {18},

pages = {6898-901},

abstract = {Single-molecule free solution hydrodynamic separation (SML-FSHS) cohesively integrates cylindrical illumination confocal spectroscopy with free solution hydrodynamic separation. This technique enables single-molecule analysis of size separated DNA with 100% mass detection efficiency, high sizing resolution and wide dynamic range, surpassing the performance of single molecule capillary electrophoresis. Furthermore, SML-FSHS required only a bare fused silica microcapillary and simple pressure control rather than complex high voltage power supplies, sieving matrices, and wall coatings. The wide dynamic range and high sizing resolution of SML-FSHS was demonstrated by separating both large DNA (23 vs 27 kbp) and small DNA (100 vs 200 bp) under identical conditions. Separations were successfully performed with near zero sample consumption using as little as 5 pL of sample and 240 yoctomoles ( approximately 150 molecules) of DNA. Quantitative accuracy was predominantly limited by molecular shot noise. Furthermore, the ability of this method to analyze of single molecule nanosensors was investigated. SML-FSHS was used to examine the thermodynamic equilibrium between stochastically open molecular beacon and target-bound molecular beacon in the detection of E. coli 16s rRNA targets.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3235,

title = {A surface topography assisted droplet manipulation platform for biomarker detection and pathogen identification},

author = { Y. Zhang and S. Park and K. Liu and J. Tsuan and S. Yang and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/21046055},

doi = {10.1039/c0lc00296h},

issn = {1473-0189 (Electronic) 

1473-0189 (Linking)},

year  = {2011},

date = {2011-02-07},

journal = {Lab Chip},

volume = {11},

number = {3},

pages = {398-406},

abstract = {This paper reports a droplet microfluidic, sample-to-answer platform for the detection of disease biomarkers and infectious pathogens using crude biosamples. The platform exploited the dual functionality of silica superparamagnetic particles (SSP) for solid phase extraction of DNA and magnetic actuation. This enabled the integration of sample preparation and genetic analysis within discrete droplets, including the steps of cell lysis, DNA binding, washing, elution, amplification and detection. The microfluidic device was self contained, with all reagents stored in droplets, thereby eliminating the need for fluidic coupling to external reagent reservoirs. The device incorporated unique surface topographic features to assist droplet manipulation. Pairs of micro-elevations were created to form slits that facilitated efficient splitting of SSP from droplets. In addition, a compact sample handling stage, which integrated the magnet manipulator, the droplet microfluidic device and a Peltier thermal cycler, was built for convenient droplet manipulation and real-time detection. The feasibility of the platform was demonstrated by analysing ovarian cancer biomarker Rsf-1 and detecting Escherichia coli with real time polymerase chain reaction and real time helicase dependent amplification.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3236,

title = {An all-in-one microfluidic device for parallel DNA extraction and gene analysis},

author = { Y. Zhang and S. Park and S. Yang and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/20632111},

doi = {10.1007/s10544-010-9458-6},

issn = {1572-8781 (Electronic) 

1387-2176 (Linking)},

year  = {2010},

date = {2010-12-01},

journal = {Biomed Microdevices},

volume = {12},

number = {6},

pages = {1043-9},

abstract = {We have developed a microfluidic device capable of fully integrated sample preparation and gene analysis from crude biosamples such as whole blood. Our platform takes the advantage of the silica superparamagnetic particle based solid phase extraction to develop an all-in-one scheme that performs cell lysis, DNA binding, washing, elution and the PCR in the same reaction chamber. The device also employs a unique reagent loading scheme, allowing efficient preparation of multiple reactions via a single injection channel. In addition, PCR is performed in a droplet-in-oil manner, eliminating the need for chamber sealing. The combination of these design features greatly reduces the complexity in implementing fully integrated lab-on-a-chip systems for genetic detection, facilitating parallel analysis of multiple samples or genes on a single microchip. The capability of the device is demonstrated by performing DNA isolation from the human whole blood sample and analyzing the Rsf-1 gene using the TaqMan probe based gene specific PCR assays.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3239,

title = {DNA methylation detection using MS-qFRET, a quantum dot-based nanoassay},

author = { V. J. Bailey and B. P. Keeley and C. R. Razavi and E. Griffiths and H. E. Carraway and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/20362674},

doi = {10.1016/j.ymeth.2010.03.007},

issn = {1095-9130 (Electronic) 

1046-2023 (Linking)},

year  = {2010},

date = {2010-11-01},

journal = {Methods},

volume = {52},

number = {3},

pages = {237-41},

abstract = {Detection of aberrant promoter hypermethylation of tumor suppressor genes can be used as a prognostic or predictive marker for carcinogenesis. Since epigenetic modifying agents are FDA approved for treatment of patients with myelodysplastic syndrome, laboratory correlative tools to monitor response to this targeted therapy are important. Methylation specific quantum dot fluorescence resonance energy transfer (MS-qFRET) is a nanotechnology assay that enables the detection of methylation and its changes in a sensitive, quantifiable manner. It utilizes quantum dot-mediated fluorescence resonance energy transfer to achieve highly sensitive detection of DNA methylation. Template DNA is first treated with sodium bisulfite such that unmethylated cytosines are converted to uracil while methylated cytosines remain unconverted. Thereafter, the converted template is amplified using biotinylated methylation-specific primers. Quantum dots, functionalized with streptavidin, serve both as a scaffold to capture amplicons and as a donor for transferring energy to the Cy5 acceptor that is incorporated into the amplicons during PCR. Thus, the status of DNA methylation can be determined according to the level of FRET. In this report, MS-qFRET is validated in cell lines and then used to detect the status of p15(INK4B) methylation in clinical samples from eight patients with acute myeloid leukemia.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN5136,

title = {Quantum Dots DNA Nanosensors - Ultrasensitive Platform for Detecting Genomic Cancer Markers},

author = { T. H. Wang and V.J. Bailey and Y. Zhang and K. J. Liu},

year  = {2010},

date = {2010-07-26},

journal = {BIOforum Europe, 1-2:28-30},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3237,

title = {Dual-sensitive micellar nanoparticles regulate DNA unpacking and enhance gene-delivery efficiency},

author = { X. Jiang and Y. Zheng and H. H. Chen and K. W. Leong and T. H. Wang and H. Q. Mao},

url = {https://www.ncbi.nlm.nih.gov/pubmed/20440698},

doi = {10.1002/adma.200903933},

issn = {1521-4095 (Electronic) 

0935-9648 (Linking)},

year  = {2010},

date = {2010-06-18},

journal = {Adv Mater},

volume = {22},

number = {23},

pages = {2556-60},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN5135,

title = {Transforming Microfluidics into Laboratory Automation},

author = { Tza-Huei Wang and Pak Kin Wong},

url = {http://jla.sagepub.com/content/15/3/A15.short},

doi = {10.1016/j.jala.2010.03.002},

issn = {15355535},

year  = {2010},

date = {2010-06-01},

journal = {Journal of the Association for Laboratory Automation},

volume = {15},

number = {3},

pages = {A15-A16},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3240,

title = {Single-tube analysis of DNA methylation with silica superparamagnetic beads},

author = { V. J. Bailey and Y. Zhang and B. P. Keeley and C. Yin and K. L. Pelosky and M. Brock and S. B. Baylin and J. G. Herman and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/20360128},

doi = {10.1373/clinchem.2009.140244},

issn = {1530-8561 (Electronic) 

0009-9147 (Linking)},

year  = {2010},

date = {2010-06-01},

journal = {Clin Chem},

volume = {56},

number = {6},

pages = {1022-5},

abstract = {BACKGROUND: DNA promoter methylation is a signature for the silencing of tumor suppressor genes. Most widely used methods to detect DNA methylation involve 3 separate, independent processes: DNA extraction, bisulfite conversion, and methylation detection via a PCR method, such as methylation-specific PCR (MSP). This method includes many disconnected steps with associated losses of material, potentially reducing the analytical sensitivity required for analysis of challenging clinical samples. METHODS: Methylation on beads (MOB) is a new technique that integrates DNA extraction, bisulfite conversion, and PCR in a single tube via the use of silica superparamagnetic beads (SSBs) as a common DNA carrier for facilitating cell debris removal and buffer exchange throughout the entire process. In addition, PCR buffer is used to directly elute bisulfite-treated DNA from SSBs for subsequent target amplifications. The diagnostic sensitivity of MOB was evaluated by methylation analysis of the CDKN2A [cyclin-dependent kinase inhibitor 2A (melanoma, p16, inhibits CDK4); also known as p16(INK4a)] promoter in serum DNA of lung cancer patients and compared with that of conventional methods. RESULTS: Methylation analysis consisting of DNA extraction followed by bisulfite conversion and MSP was successfully carried out within 9 h in a single tube. The median pre-PCR DNA yield was 6.61-fold higher with the MOB technique than with conventional techniques. Furthermore, MOB increased the diagnostic sensitivity in our analysis of the CDKN2A promoter in patient serum by successfully detecting methylation in 74% of cancer patients, vs the 45% detection rate obtained with conventional techniques. CONCLUSIONS: The MOB technique successfully combined 3 processes into a single tube, thereby allowing ease in handling and an increased detection throughput. The increased pre-PCR yield in MOB allowed efficient, diagnostically sensitive methylation detection.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3238,

title = {Decoding circulating nucleic acids in human serum using microfluidic single molecule spectroscopy},

author = { K. J. Liu and M. V. Brock and M. Shih Ie and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/20364832},

doi = {10.1021/ja100342q},

issn = {1520-5126 (Electronic) 

0002-7863 (Linking)},

year  = {2010},

date = {2010-04-28},

journal = {J Am Chem Soc},

volume = {132},

number = {16},

pages = {5793-8},

abstract = {Circulating nucleic acid (CNA) has been the focus of recent research as a noninvasive source of biomarker candidates. Among these markers, DNA fragment size has shown promise for discerning the source of CNA molecules in cancer and prenatal diagnostics. We have developed a one-step assay for analyzing circulating DNA size and quantity directly in human serum. Microfluidic cylindrical illumination confocal spectroscopy and fluorescence burst size analysis are used to individually count and size fluorescently-labeled CNA molecules as they are driven through a microfluidic constriction. First, single molecule sizing was performed on lambda Hind III digest DNA to obtain a size calibration curve. A linear relation between DNA length and burst size was seen from 564 bp to 27.5 kbp. Subsequently, the single molecule assay parameters were optimized. Finally, DNA sizing analysis was performed on serum samples from both early and late stage lung cancer patients. This assay was performed directly in patient serum using only a single reagent, a simple DNA intercalating dye, and without the need for DNA isolation or enzymatic amplification steps. This demonstrates that microfluidic single molecule spectroscopy can be a rapid, facile, and inexpensive alternative to the established PCR-based methods that have been used near exclusively for CNA analysis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3243,

title = {Counting single molecules in sub-nanolitre droplets},

author = { T. D. Rane and C. M. Puleo and K. J. Liu and Y. Zhang and A. P. Lee and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/20066242},

doi = {10.1039/b917503b},

issn = {1473-0197 (Print) 

1473-0189 (Linking)},

year  = {2010},

date = {2010-01-21},

journal = {Lab Chip},

volume = {10},

number = {2},

pages = {161-4},

abstract = {We demonstrate single biomolecule detection and quantification within sub-nanolitre droplets through application of Cylindrical Illumination Confocal Spectroscopy (CICS) and droplet confinement within a retractable microfluidic constriction.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3245,

title = {Enzymatic incorporation of multiple dyes for increased sensitivity in QD-FRET sensing for DNA methylation detection},

author = { V. J. Bailey and B. P. Keeley and Y. Zhang and Y. P. Ho and H. Easwaran and M. V. Brock and K. L. Pelosky and H. E. Carraway and S. B. Baylin and J. G. Herman and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/19904794},

doi = {10.1002/cbic.200900630},

issn = {1439-7633 (Electronic) 

1439-4227 (Linking)},

year  = {2010},

date = {2010-01-04},

journal = {Chembiochem},

volume = {11},

number = {1},

pages = {71-4},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3242,

title = {Engineering an artificial alveolar-capillary membrane: a novel continuously perfused model within microchannels},

author = { D. D. Nalayanda and Q. Wang and W. B. Fulton and T. H. Wang and F. Abdullah},

url = {https://www.ncbi.nlm.nih.gov/pubmed/20105578},

doi = {10.1016/j.jpedsurg.2009.10.008},

issn = {1531-5037 (Electronic) 

0022-3468 (Linking)},

year  = {2010},

date = {2010-01-01},

journal = {J Pediatr Surg},

volume = {45},

number = {1},

pages = {45-51},

abstract = {INTRODUCTION: Pulmonary hypoplasia is a condition of the newborn that is characterized by underdeveloped lungs and poor outcome. One strategy in the treatment of patients with hypoplasia is to augment underdeveloped lungs using biocompatible artificial lung tissue. However, one central challenge in current pulmonary tissue engineering efforts remains the development of a stable bio-mimetic alveolar-capillary membrane. Accordingly, we have built a series of bio-mimetic microfluidic devices that specifically model the alveolar-capillary membrane. Current designs include a single-layer microchip that exposes alveolar and endothelial cell types to controlled fluidic stimuli. A more advanced multi-layered device allows for alveolar cells to be cultured at an air interface while allowing constant media nourishment and waste removal, thus better mimicking the physiologic milieu of the alveolar-capillary interface. Both devices possess the benefit of parallel testing. MATERIAL AND METHODS: Microdevices were fabricated using soft lithography in a biocompatible transparent polymeric material, polydimethyl siloxane, sealed covalently to glass. The multistage microdevice also integrated a suspended polyethylene terephthalate membrane connected via microfluidic channels to constant media and air access. Pulmonary endothelial (HMEC-1) and alveolar epithelial (A549) cell lines, along with fetal pulmonary cells (FPC) harvested from Swiss Webster mice at day 18 gestational age, were studied under multiple hydrodynamic shear conditions and liquid-to-cell ratio regimes. Cultures were examined for cell viability, function and proliferation to confluent monolayers. A549 cells cultured at an air-interface in a microdevice was also tested for their ability to maintain cell phenotype and function. RESULTS: The single-layer differential flow microdevice allowed for a systematic determination of the optimal growth conditions of various lung-specific cell types in a microfluidic environment. Our device showed a greater surfactant based decrease in surface tension of the alveolar hypophase in A549 cultures exposed to air as compared to submerged cultures. CONCLUSIONS: We have successfully developed biomimetic microfluidic devices that specifically allow stable alveolar cell growth at the air-liquid interface. This work serves prerequisite towards an implantable artificial alveolar membrane.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3244,

title = {Quantum dots in molecular detection of disease},

author = { V. J. Bailey and C. M. Puleo and Y. P. Ho and H. C. Yeh and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/19965019},

doi = {10.1109/IEMBS.2009.5334553},

issn = {1557-170X (Print) 

1557-170X (Linking)},

year  = {2009},

date = {2009-12-08},

journal = {Conf Proc IEEE Eng Med Biol Soc},

volume = {2009},

pages = {4089-92},

abstract = {The unique photophysical properties of semiconductor quantum dots (QDs) have made them ideal for use as spectral labels and luminescent probes. In this review, applications are presented in which QDs function as active participants in nanoscale biosensor assemblies, where replacing traditional molecular fluorophores results in improved assay performance. Specific focus is on disease detection with applications including multiplexed target detection, mutation detection by coincidence analysis and QD-based FRET reporters for miRNA detection and DNA methylation analysis.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3246,

title = {Integration and application of vitrified collagen in multilayered microfluidic devices for corneal microtissue culture},

author = { C. M. Puleo and W. McIntosh Ambrose and T. Takezawa and J. Elisseeff and T. H. Wang},

url = {https://www.ncbi.nlm.nih.gov/pubmed/19865728},

doi = {10.1039/b908332d},

issn = {1473-0197 (Print) 

1473-0189 (Linking)},

year  = {2009},

date = {2009-11-21},

journal = {Lab Chip},

volume = {9},

number = {22},

pages = {3221-7},

abstract = {This paper describes the fabrication and application of microfluidic devices containing collagen vitrigel (CV) used as both a functional and sacrificial cell growth substrate for the development of corneal microtissue patches. Within the device, vacuum fixation of the CV in a dehydrated state enables quick integration with standard multilayer soft lithographic techniques, while on-chip rehydration results in a gel-like collagen substrate for microfluidic cell culture. Fluidic connectivity to both the apical and basal side of the CV permits bilayered culture of epithelium and supporting stromal cell layers. In addition, microfluidic introduction of a collagenase etching media enables sacrificial degradation of the supporting CV membrane for development of barrier tissue constructs containing minimal synthetic substrate. The utility of this platform was evaluated by miniaturizing the standard transepithelial permeability (TEP) assay in order to measure the integrity of an array of corneal tissue micropatches.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}

@article{RN3248,

title = {An open-access microfluidic model for lung-specific functional studies at an air-liquid interface},

author = { D. D. Nalayanda and C. Puleo and W. B. Fulton and L. M. Sharpe and T. H. Wang and F. Abdullah},

url = {https://www.ncbi.nlm.nih.gov/pubmed/19484389},

doi = {10.1007/s10544-009-9325-5},

issn = {1572-8781 (Electronic) 

1387-2176 (Linking)},

year  = {2009},

date = {2009-10-01},

journal = {Biomed Microdevices},

volume = {11},

number = {5},

pages = {1081-9},

abstract = {In an effort to improve the physiologic relevance of existing in vitro models for alveolar cells, we present a microfluidic platform which provides an air-interface in a dynamic system combining microfluidic and suspended membrane culture systems. Such a system provides the ability to manipulate multiple parameters on a single platform along with ease in cell seeding and manipulation. The current study presents a comparison of the efficacy of the hybrid system with conventional platforms using assays analyzing the maintenance of function and integrity of A549 alveolar epithelial cell monolayer cultures. The hybrid system incorporates bio-mimetic nourishment on the basal side of the epithelial cells along with an open system on the apical side of the cells exposed to air allowing for easy access for assays.},

keywords = {},

pubstate = {published},

tppubtype = {article}

}