Nanofluidic Platform Research Articles

Non-Negligible Diffusio-Osmosis Inside an Ion Concentration Polarization Layer

The first experimental and theoretical evidence was provided for the non-negligible role of a diffusio-osmosis in the ion concentration polarization (ICP) layer, which had been reported to be in a high Peclet number regime. Under the assumption that the hydrated shells of cations were stripped out with the amplified electric field inside the ICP layer, its concentration profile possessed a steep concentration gradient at the stripped location. Since the concentration gradient drove a strong diffusio-osmosis, the combination of electro-osmotic and diffusio-osmotic slip velocity had a form of an anomalous nonmonotonic function with both a single- and multiple-cationic solution. A direct measurement of electrolytic concentrations around the layer quantitatively validated our new investigations. This non-negligible diffusio-osmotic contribution in a micro- and nanofluidic platform or porous medium would be essential for clarifying the fundamental insight of nanoscale electrokinetics as well as guiding the engineering of ICP-based electrochemical systems.

Abstract 4893: High-throughput mutation sequencing of the full exon regions in BRCA1 and BRCA2 genes using nanofluidic-PCR prepared libraries

Abstract Breast cancer is one of the most common cancers and also the principal cause of cancer-related death among women in the world. BRCA1 and BRCA2 genes contribute to DNA repair and transcriptional regulation in response to DNA damage. Mutations of BRCA1/2 genes greatly increase lifetime risk to develop breast cancer and these mutations are frequently observed in hereditary breast cancer. In addition, recent studies suggested that breast cancer patients who carry a BRCA1 or BRCA2 germline mutation benefit from poly (ADP-ribose) polymerase (PARP) inhibitors. Therefore, it would be of great interest to develop a high-throughput approach for mutation screening of the full exon regions in BRCA1 and BRCA2 gene. We have developed a targeted gene exon-sequencing approach using a nanofluidic PCR platform, the Access Array™ system, which enables simultaneous amplification of 48 genetic regions (amplicons) from 48 samples in parallel. We have applied this system to generate the libraries of BRCA1 and BRCA2 coding regions. Total 120 target-specific primer pairs were designed covering all 22 exons of BRCA1 and 26 exons of BRCA2. A primer design strategy was applied to incorporate sample-specific barcodes and Illumina sequencing adaptors into each amplicon, allowing direct sequencing without further library preparation. The exon regions of these 2 genes have been amplified on Access Arrays using 48 multiplex mixes of the 120 primer sets in paired tumor/normal tissues from 144 breast cancer patients. We will present the next-generation sequencing data of BRCA1 and BRCA2 mutation screening collected from amplicon libraries generated using the Access Array system. Our results indicate the excellent utilization of this approach in the mutation screening of BRCA1 and BRCA2 in clinical samples. Note: This abstract was not presented at the meeting. Citation Format: Jun Wang, Guoli Li, Ming Chen, Jianfu Heng, Xinwu Guo, Limin Peng, Fan Zhang, Zibo Li, Shouman Wang, Zhi Xiao, Lizhong Dai, Wenjun Yi, Lili Tang. High-throughput mutation sequencing of the full exon regions in BRCA1 and BRCA2 genes using nanofluidic-PCR prepared libraries. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4893. doi:10.1158/1538-7445.AM2015-4893

Abstract 293: High-throughput methylation sequencing of targeted genes in breast cancer specimens using nanofluidic PCR prepared libraries

Abstract Breast cancer is a global public health issue as it is the most frequently diagnosed malignancy in women in the Western world with over a million new cases every year worldwide. Previous epigenetic analyses have identified aberrant DNA methylation signatures associated with breast cancer. Methylation alterations resulting in aberrant gene expression are key contributors to breast tumorigenesis. There are more than 100 candidate genes reported throughout the literatures as promoter hypermethylated in breast cancers (Pubmeth web resource). More recently, with the application of genome wide array-based methylation profile analysis and next generation sequencing, hundreds of new methylation candidate genes have been reported to be associated with breast cancer. It is crucial to develop an approach for targeted methylaton resequencing to validate these candidate genes in hundreds of patient samples and develop a breast cancer detection panel for early diagnostics. We have developed a targeted gene bisulfite-sequencing approach using a nanofluidic platform, the Access Array™ system, which enables simultaneous amplification of 48 genetic regions (amplicon) from 48 samples in parallel. We have selected 48 breast cancer associated methylation candidate genes, each commonly reported by multiple literatures. Bisulfite-specific-sequencing (BSP) primers were designed for the promoter regions of these genes. In addition, we have applied a primer design strategy that incorporates sample-specific barcodes and Illumina (Miseq) sequencing adaptors into each amplicon, removing the need for additional library preparation before sequencing. We will present methylation sequencing data collected from amplicon libraries generated using the Access Array system. The 48 promoter regions have been amplified in paired tumor/normal tissues and plasma DNA from 48 breast cancer patients. Our results indicate the excellent utilization of this approach in the validation of methylation biomarkers and selection of detection panel for breast cancer. Citation Format: Jun Wang, Zibo Li, Yepeng Wu, Xinwu Guo, Shengyun Li, Zhi Xiao, Feiyu Chen, Zhongping Deng, Lizhong Dai, Wenjun Yi, Lili Tang. High-throughput methylation sequencing of targeted genes in breast cancer specimens using nanofluidic PCR prepared libraries. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 293. doi:10.1158/1538-7445.AM2014-293

Dielectrophoretic separation of bioparticles in microdevices: A review

In recent years, dielectrophoretic force has been used to manipulate colloids, inert particles, and biological microparticles, such as red blood cells, white blood cells, platelets, cancer cells, bacteria, yeast, microorganisms, proteins, DNA, etc. This specific electrokinetic technique has been used for trapping, sorting, focusing, filtration, patterning, assembly, and separating biological entities/particles suspended in a buffer medium. Dielectrophoretic forces acting on particles depend on various parameters, for example, charge of the particle, geometry of the device, dielectric constant of the medium and particle, and physiology of the particle. Therefore, to design an effective micro-/nanofluidic separation platform, it is necessary to understand the role of the aforementioned parameters on particle motion. In this paper, we review studies particularly related to dielectrophoretic separation in microfluidic devices. Both experimental and theoretical works by several researchers are highlighted in this article covering AC and DC DEP. In addition, AC/DC DEP, which uses a combination of low frequency AC and DC voltage to manipulate bioparticles, has been discussed briefly. Contactless DEP, a variation of DC DEP in which electrodes do not come in contact with particles, has also been reviewed. Moreover, dielectrophoretic force-based field flow fractionations are featured to demonstrate the bioparticle separation in microfluidic device. In numerical front, a comprehensive review is provided starting from the most simplified effective moment Stokes-drag (EMSD) method to the most advanced interface resolved method. Unlike EMSD method, recently developed advanced numerical methods consider the size and shape of the particle in the electric and flow field calculations, and these methods provide much more accurate results than the EMSD method for microparticles.

A Simple, Single-Carbon-Nanotube Nanofludic Platform for Fundamental Transport Studies

Fundamental understanding of ionic and molecular transport phenomena in a simple model nanopore is critical for elucidating function mechanisms of much more complex biological systems, and for advancing technological areas such as membrane separation, energy harvesting/storage, and single molecule detection. For this goal, carbon nanotubes (CNTs) offer key advantages as model nanofluidic channels due to their simple chemical composition and structure (known with atomic precision), robustness, facile length and diameter control, and straightforward local functionalization at their open rim. CNTs have also very interesting fluidics properties such as enhanced pressure-driven fluid transport rates, unusually high electroosmotic flow, and ionic selectivity.Here, we present our work toward developing and validating a novel nanofluidic platform featuring an individual carbon nanotube (CNT) as the flow channel in an advanced Coulter Counter. To fabricate the CNT nanofluidic device, vertically aligned single-walled CNTs are synthesized directly on a suspended silicon nitride membrane and then bound in a solid matrix before an individual CNT is opened by focused ion beam milling. Single-molecule translocation studies with small molecular size analytes suggest the successful fabrication of a Coulter Counter with a-few-nm wide CNT nanochannel. Our initial ionic conductivity studies indicate a power-law increase of conductance with KCl concentration in CNT channels, a dependence that seems to be unique of CNT pores.

Multiplex Screening for Blood-Borne Viral, Bacterial, and Protozoan Parasites using an OpenArray Platform

The use of nucleic acid tests for detection of pathogens has improved the safety of blood products. However, ongoing pathogen emergence demonstrates a need for development of devices testing for multiple pathogens simultaneously. One approach combines two proven technologies: Taqman chemistry for target identification and quantification and the OpenArray nanofluidic real-time PCR platform for spatial multiplexing of assays. A panel of Taqman assays was developed to detect nine blood-borne pathogens (BBPs): four viral, two bacterial, and three protozoan parasites. The custom BBP OpenArray plate with 18 assays was tested for specificity and analytical sensitivity for nucleic acid from each purified pathogen and with pathogen-spiked human blood and plasma samples. For most targets, the limits of detection (10 to 10,000 copies/mL) were comparable with existing real-time platforms. The testing of the BBP OpenArray with pathogen-spiked coded human plasma or blood samples and negative control specimens demonstrated no false-positive results among the samples tested and correctly identified pathogens with the lowest concentration detected ranging from 10 cells/mL (Trypanosoma cruzi) to 10,000 cells/mL (Escherichia coli). These results represent a proof of concept that indicated the BBP OpenArray platform in combination with Taqman chemistry may provide a multiplex real-time PCR pathogen detection method that points the way for a next-generation platform for infectious disease testing in blood.

Abstract 3997: Nanoproteomic analysis of extracellular receptor kinase-1/2 post-translational activation in microdissected human hyperplastic aberrant crypt foci.

Abstract Oncogenic activation resulting in hyperproliferative lesions within the colonic mucosa has been identified in putative precancerous lesions, aberrant crypt foci (ACF). KRAS (codons 12 and 13) and BRAFV600E mutation status was determined in 172 ACF identified in colorectum of screening subjects by in situ high-definition, magnifying chromoendoscopy. Lesions were stratified according to histology: serrated hyperplastic versus distended (non-serrated) hyperplastic. Due to their limiting size, however, it was not technically feasible to examine downstream signaling consequences of these oncogenic mutations. We have combined ultraviolet-infrared (UV/IR) micro-dissection with an ultra-sensitive nanofluidic proteomic immunoassay (NIA) to enable accurate quantification of post-translational modifications to mitogen-activated protein kinase (MAPK) in total protein lysates isolated from hyperproliferative crypts and adjacent normal mucosa. Using this approach, levels of singly and dually (activated) phosphorylated isoforms of extracellular receptor kinase(ERK)-1 and -2 were quantified in samples containing as little as 16-ng of total protein recovered from <200 colon epithelial cells. ERK activation is responsible for the observed hyperplasia found in these early lesions, but its activation state is not directly dependent on KRAS and/or BRAF mutation status. This study describes the novel use of a sensitive nanofluidic platform to measure oncogene-driven proteomic changes in diminutive lesions procured at the time of endoscopy and highlights the advantage of this approach over classical immunohistochemistry-based analyses. Citation Format: David A. Drew, Thomas Devers, Nicole Horelik, Shi Yang, Michael O'Brien, Rong Wu, Daniel W. Rosenberg. Nanoproteomic analysis of extracellular receptor kinase-1/2 post-translational activation in microdissected human hyperplastic aberrant crypt foci. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3997. doi:10.1158/1538-7445.AM2013-3997

Nanoproteomic analysis of extracellular receptor kinase‐1/2 post‐translational activation in microdissected human hyperplastic colon lesions

Oncogenic activation resulting in hyperproliferative lesions within the colonic mucosa has been identified in putative precancerous lesions, aberrant crypt foci (ACF). KRAS and BRAF mutation status was determined in 172 ACF identified in the colorectum of screening subjects by in situ high-definition, magnifying chromoendoscopy. Lesions were stratified according to histology (serrated vs. distended). Due to their limiting size, however, it was not technically feasible to examine downstream signaling consequences of these oncogenic mutations. We have combined ultraviolet-infrared (UV/IR) microdissection with an ultrasensitive nanofluidic proteomic immunoassay (NIA) to enable accurate quantification of posttranslational modifications to mitogen-activated protein kinase (MAPK) in total protein lysates isolated from hyperproliferative crypts and adjacent normal mucosa. Using this approach, levels of singly and dually (activated) phosphorylated isoforms of extracellular receptor kinase(ERK)-1 and ERK-2 were quantified in samples containing as little as 16 ng of total protein recovered from <200 cells. ERK activation is responsible for observed hyperplasia found in these early lesions, but is not directly dependent on KRAS and/or BRAF mutation status. This study describes the novel use of a sensitive nanofluidic platform to measure oncogene-driven proteomic changes in diminutive lesions and highlights the advantage of this approach over classical immunohistochemistry-based analyses.

Nanofluidic Digital PCR for KRAS Mutation Detection and Quantification in Gastrointestinal Cancer

Concomitant quantification of multiple mutant KRAS (v-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog) alleles may provide information in addition to that provided by standard mutation-detection procedures. We assessed the feasibility of a nanofluidic digital PCR array platform to detect and quantify KRAS mutations simultaneously in clinically relevant samples. We assessed 2 groups of patients (colorectal and pancreatic disease): Group 1 consisted of 27 patients with colorectal carcinomas, 14 patients with adenomas, and 5 control individuals; group 2 consisted of 42 patients with pancreatic carcinoma, 4 with adenocarcinomas of the ampulla, and 6 with chronic pancreatitis). Digital PCR was performed with the Digital Array Chip (Fluidigm). Nanofluidic digital PCR detected mutant alleles at 0.05% to 0.1%, depending on the variant analyzed. For the colorectal disease group, conventional PCR detected 9 (64%) of 14 adenomas that were positive for KRAS mutants, whereas digital PCR increased this number to 11 (79%) of 14. Sixteen (59%) of 27 carcinomas showed KRAS mutation with conventional PCR. Two additional cases were detected with digital PCR. In 5 cases (3 adenomas, 2 carcinomas), the total number of mutant alleles changed. For the pancreatic disease group, digital PCR increased the number of positive cases from 26 to 34 (81%) and identified ≥ 2 mutant alleles in 25 cases, compared with conventional PCR, which identified multiple KRAS mutant alleles in only 12 cases. A good correlation was observed between results obtained with tumor biopsies and those obtained with pancreatic juice. Digital PCR provides a robust, quantitative measure of the proportion of KRAS mutant alleles in routinely obtained samples. It also allows a better classification of tumors, with potential clinical relevance.

Real-time analysis and selection of methylated DNA by fluorescence-activated single molecule sorting in a nanofluidic channel

Epigenetic modifications, such as DNA and histone methylation, are responsible for regulatory pathways that affect disease. Current epigenetic analyses use bisulfite conversion to identify DNA methylation and chromatin immunoprecipitation to collect molecules bearing a specific histone modification. In this work, we present a proof-of-principle demonstration for a new method using a nanofluidic device that combines real-time detection and automated sorting of individual molecules based on their epigenetic state. This device evaluates the fluorescence from labeled epigenetic modifications to actuate sorting. This technology has demonstrated up to 98% accuracy in molecule sorting and has achieved postsorting sample recovery on femtogram quantities of genetic material. We have applied it to sort methylated DNA molecules using simultaneous, multicolor fluorescence to identify methyl binding domain protein-1 (MBD1) bound to full-duplex DNA. The functionality enabled by this nanofluidic platform now provides a workflow for color-multiplexed detection, sorting, and recovery of single molecules toward subsequent DNA sequencing.

Travels in time: Assessing the functional complexity of T cells

When the host encounters a pathogen, the ensuing immune response involves a complex set of cellular responses distributed across many different types of cells. In T and B lymphocytes of the adaptive immune system, these responses include irreversible differentiation events that generate functionally specialized subpopulations of cells (1). Understanding how pathogens and vaccines influence the number, type, and efficacy of specific differentiation states in the T-cell compartment is a major goal in immunology. The study by Han et al. in PNAS (2) interrogates the functional response of individual T cells over time using a nanofluidic platform. Their experiments reveal that the sequence of cytokines released over time by individual activated T cells is highly diverse but tightly programmed. Their work suggests that understanding the complexity of the T-cell response may not only be a matter of cataloging the possible phenotypes present in a cross-section of the T-cell population but will also need to involve understanding how those functions change over time.

Long Term Single Molecule TIRF Observation of Biomolecules without Immobilization

Single-molecules fluorescence techniques have yielded valuable structural and dynamical information about molecular biological machines. The most common approaches used to observe single molecules in biology are confocal spectroscopy of freely diffusing molecules and total internal reflection (TIR) microscopy of surface attached molecules. With the latter technique it is possible to perform long-term observation of individual molecules, but at the cost of complex immobilization procedures that cannot be generally applied and often perturb the sample. Here we demonstrate a versatile technology that combines the advantages of both existing approaches, i.e. freely diffusing molecules and long-term observation with high signal to noise, in a simple, easy to handle, low-cost nanofluidic platform. Biomolecules flow through channels that are less than 100 nm deep, which keeps them within the TIR field, as required to fully exploit the high signal to noise possible with this optical sectioning technique. Rather than employing typical expensive nanofluidic technology, the nanochannels are generated using controlled channel collapse on a microfluidic device made from PDMS, ensuring easy fabrication and handling. We demonstrate that biomolecules labeled with single fluorophores can be imaged with millisecond time-resolution for several seconds. In addition, the design of the control channel of the device makes it possible to achieve high fluorophore photostability by removing oxygen without any need to add chemicals. By having a continuous stream of biomolecules flowing through the nanochannels, thousands of biomolecules can be measured each hour, illustrating the potential for automated experiments. We demonstrate the compatibility of the device with large biological complexes by showing multi-channel imaging of freely diffusing single nucleosomes.

Soft Matter Based Nanofluidic Platform for Highly Parallel Mixing of Sub-Attoliter Total Volumes

Small unilamellar lipid vesicles (SUVs) arrayed at solid supports are useful as nanoscopic test tubes for microscopy-based experiments with few or single molecules(1). Encapsulation in SUVs enables spatial confinement of biomolecules at a glass coverslip while retaining them free in solution and in a native-like environment. Manipulating and mixing the content of individual SUVs has, however, proven challenging due to their nanoscopic dimensions. We demonstrate a fluidic platform that allows triggering highly parallelized mixing of reagents loaded in individual SUVs with self-enclosed volumes as small as 10−19 l(2). We encapsulated one set of reactants in vesicles immobilized at a glass surface and triggered fusion of these stationary nanoreactors with diffusing vesicles of opposite charge that carried a complementary set of reactants. Productive and leakage-free mixing was achieved on ∼85% of the ∼106 cm−2 surface-tethered vesicles with up to 3-4 consecutive injections per reactor. Development of ultra-small-volume fluidic platforms will enable novel ways to implement simultaneous screening of biochemical properties, molecular function or confined reactions over millions of samples while consuming total reagent volumes of few picoliters.

Abstract 310: BCR-ABL1 transcript detection in patients with Philadelphia-positive Acute Lymphoblastic Leukemia (ALL) using a new and high sensitive nanofluidic method

Abstract Background: Detection of residual leukemic cells by measuring BCR-ABL1 transcript level with assays based on quantitative polymerase chain reaction (qPCR) is necessary in the monitoring of minimal residual disease in patients with BCR-ABL1 positive ALL. Aim: To investigate the efficacy of a high sensitive method based on nanofluidic platform (Fluidigm Corporation, South San Francisco, CA) to detect and quantify residual and rare BCR-ABL1 copies, we compared results obtained by conventional qPCR with those obtained by the nanofluidic approach. Patients and methods: We analyzed 60 BCR-ABL1 positive ALL samples expressing p190 (42) or p210 (18) isoform, first by TaqMan absolute qPCR (Applied Biosystems 7900HT Fast Real-Time PCR System). BCR-ABL1 target gene and ABL1 control gene copies were derived by the interpolation of cycle threshold values to the appropriate standard curve obtained using serial plasmid dilutions containing known BCR-ABL1 or ABL1 gene copies. Results: Samples resulted in complete (22) or major (38) molecular response (BCR-ABL1/ABL1 ratio ≪ 0.001 or &amp;lt; 0.1, respectively; 3,8 BCR-ABL1 mean copy number copies). Subsequently we reanalyzed the samples using the 12.765 Digital array (Fluidigm) and the integrated BioMark Digital PCR Analysis software (Fluidigm). The nanofluidic biochip consists in twelve panels, each containing 765 individual reaction chambers where samples are portioned prior to qPCR. As fluorescent signal is produced only in chambers containing copies of the target sequence, digital array provides an absolute quantification by counting the number of positive reactions. Sensitivity and reproducibility of the assay were assessed using six serial dilutions of plasmids (Ipsogen) expressing known BCR-ABL1 p190 transcript copy number (10000; 1000; 100; 50; 10; 1 copies). A 2 µl volume of input cDNA was loaded and two panel for each dilution were used. Results showed a detection rate until a copy of target sequence and a pairing significantly effective between replicates. We then analyzed duplicates of BCR-ABL1 ALL samples: digital array resulted positive in 21 of major molecular response samples (2,13 BCR-ABL1 mean copy number copies) and in any complete molecular response samples. In conclusion, the Fluidigm nanofluidic platform provides a high sensitive assay, able to detect until a single copy of BCR-ABL1 transcript, and it could provide an accurate monitoring method for BCR-ABL1-positive ALL CR patients. Supported by European LeukemiaNet, AIL, AIRC, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 310. doi:10.1158/1538-7445.AM2011-310

Use of a High Sensitive Nanofluidic Array for the Detection of Rare Copies of BCR-ABL1 Transcript In Patients with Philadelphia-Positive Acute Lymphoblastic Leukemia (ALL).

AbstractAbstract 1677The BCR-ABL1 positive ALL is the most frequent and prognostically most unfavorable subtype of ALL in adults. Treatment strategies based on tyrosine kinase inhibitors of first and second generation have improved overall treatment results, with a rapid and complete remission (CR) rate ranging 90%. Nevertheless most patients experienced hematological relapse in a short time, also after hematopoietic stem cell transplantation. Therefore, the detection of residual leukemic cells by measuring BCR-ABL1 transcript level is absolutely required to monitor minimal residual disease and to detect early relapse. Different assays based on quantitative polymerase chain reaction (qPCR) are available and they vary in respect to the type of instrument used, the primer and probe location, the real-time chemistry and the control gene used. Differences among methods can lead to a variation in the sensitivity and measurement reliability. In order, to investigate the efficacy of a high sensitive method based on nanofluidic platform (Fluidigm Corporation, South San Francisco, CA) to detect and quantify residual and rare BCR-ABL1 copies, we compared the molecular results obtained by conventional qPCR with those obtained by nanofluidic Fluidigm approach. At the time of writing, 60 BCR-ABL1-positive ALL samples (42 positive for the p190 BCR-ABL1 isoform and 18 for the p210) were analyzed. First, we performed a TaqMan absolute quantitative PCR (Applied Biosystems 7900HT Fast Real-Time PCR System); RNA integrity was evaluated using the control gene ABL1. Both BCR-ABL1 or ABL1 copy number copies were derived by the interpolation of cycle threshold (Ct) values to the appropriate standard curve obtained using different plasmid dilutions (Ipsogen), each containing known BCR-ABL1 or ABL1 gene copies. Samples resulted in complete (38.3%) or major (61.7%) molecular response (BCR-ABL1/ABL1 ratio ≤ 0.001 or < 0.1, respectively and according to the International Scale). Subsequently, we quantified again the samples using the 12.765 Digital array (Fluidigm). This is a nanofluidic biochip that consists in twelve panels, each containing 765 individual reaction chambers of 6 nL volume. Samples are portioned prior to qPCR into the single chambers of the panel; as fluorescent signal is produced only in chambers containing copies of the target sequence, digital array provides an absolute quantification by counting the number of positive reactions. Following amplification, digital raw data are processed by the BioMark Digital PCR Analysis software (Fluidigm), that estimates the true number of molecules per chamber using the Poisson probabilistic distribution. First, we assessed the sensitivity and reproducibility of the assay using six serial dilutions of plasmids (Ipsogen) expressing known copy number of BCR-ABL1 p190 transcript (10000; 1000; 100; 50; 10; 1 copies). A 2 μl volume of input leukemia cDNA was loaded and two panels for each dilution were used. Analysis parameters chosen for digital raw data processing were automated set threshold and target Ct range 20–40. Results showed a detection rate until a copy of target sequence (Fig. 1) and a pairing significantly effective between replicates. We then analyzed duplicates of BCR-ABL1 positive ALL samples with a positive control for each chip: digital array resulted positive in 22 major molecular response samples (59.46%; 3.25 median number of copies detected, range 0.5–33.5), and in 1 complete molecular response sample (4.34%). Moreover, the comparison between TaqMan and Fluidigm methods resulted in the detection of the same BCR-ABL1 copies in 53.34% (group I); in 35.0% the quantity detected by Fluidigm was lower than that detected by TaqMan (group II); finally, in 11.66% the Fluidigm approach resulted more sensitive than TaqMan (group III). In conclusion, we demonstrated that the Fluidigm nanofluidic platform provides a high sensitive assay, able to detect until a single copy of BCR-ABL1 transcript, as demonstrated with plasmid serial dilution detection rate and with samples in molecular remission and it could provide an accurate monitoring method for BCR-ABL1-positive ALL CR patients. Supported by: European LeukemiaNet, AIL, AIRC, Fondazione Del Monte di Bologna e Ravenna, FIRB 2006, PRIN 2008, Ateneo RFO grants, Project of integreted program (PIO), Programma di Ricerca Regione – Università 2007 – 2009. [Display omitted] Disclosures:Baccarani:NOVARTIS: Honoraria; BRISTOL MYERS SQUIBB: Honoraria. Martinelli:Novartis: Consultancy, Honoraria; BMS: Honoraria; Pfizer: Consultancy.

Experimental Generation of SNP Haplotype Signatures in Patients with Sickle Cell Anaemia

BackgroundSickle cell anemia is caused by a single type of mutation, a homozygous A→T substitution in the ß globin gene. Clinical severity is diverse, partially due to additional, disease-modifying genetic factors. We are studying one such modifier locus, HMIP (HBS1L-MYB intergenic polymorphism, chromosome 6q23.3). Working with a genetically admixed patient population, we have encountered the necessity to generate haplotype signatures of genetic markers to label genomic fragments with distinct genealogical origin at this locus. With the goal to generate haplotype signatures from patients experimentally, we have investigated the suitability of an existing nanofluidic assay platform to perform phase alignment with single-nucleotide polymorphism alleles.Methodology/Principal FindingsPatient DNA samples were loaded onto Fluidigm Digital Arrays and individual DNA molecules were assayed with allele-specific probes for SNP markers. Here we present data showing the utility of the nanofluidic approach, yielding haplotype data identical to those obtained with a family-based method. We then determined haplotype composition in a group of patients with sickle cell disease, including in those where a mathematical inference approach gave ambiguous or misleading results. Experimental phasing of genotypes across 3.8 kb for rs9399137, rs9402685, and rs11759553 created unequivocal haplotype signatures for each of the patients. In 68 patients, we found 8 copies of a haplotype signature (‘C-C-T’), which is known to be prevalent in Europeans but to be absent in West African populations. We have confirmed the identity of our phased allele pairs by single-molecule sequencing and have demonstrated, in principle, that three-allele phasing (using three colors) is a potential extension to this method.Conclusions/SignificancePhased haplotypes yield more information than the individual marker genotypes. Procedures such as the one described here would therefore benefit genetic mapping and functional studies as well as diagnostic procedures where the identity or parental origin of short genetic fragments is of importance.

Abstract 1147: High-throughput nanofluidic PCR-based preparation of 454 sequencing libraries for identifying mutations in EGFR and MET

Abstract The epidermal growth factor receptor (EGFR) and mesenchymal-epithelial transition factor (MET) play important roles in cancer development and are promising targets for anticancer therapies. Mutations in the EGFR and MET genes are associated with multiple types of cancer and the ability to detect variants in these genes is crucial for a better understanding of disease progression and response to targeted therapeutics. Next-generation sequencers (e.g. 454, Illumina) hold great potential for detecting disease-specific alterations within patient populations for a given cancer type. However, current approaches for targeted resequencing of specific genetic regions are not well suited to studying the hundreds or thousands of patient samples required to assign significance to mutations. We have developed a novel nanofluidic platform, the Access Array™ system, which enables the robust, simultaneous amplification of 48 PCR products (amplicon) from 48 samples in parallel using only 50ng input DNA per sample. In addition, we have developed a primer design strategy that incorporates sample-specific barcodes and 454 sequencing adaptors into each amplicon, removing the need for library preparation before sequencing. We will present sequencing data collected from amplicon libraries prepared using the Access Array system to amplify 48 exon regions of the EGFR and MET genes from the NCI-60 cell lines. Sequencing data from these libraries demonstrates excellent uniformity in representation between amplicons and across samples. We will present data mapping mutations within these genes across the NCI-60, and discuss the use of Access Array enrichment panels in monitoring mutations across large patient populations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1147.

Quantifying EGFR Alterations in the Lung Cancer Genome with Nanofluidic Digital PCR Arrays

The EGFR [epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homolog, avian)] gene is known to harbor genomic alterations in advanced lung cancer involving gene amplification and kinase mutations that predict the clinical response to EGFR-targeted inhibitors. Methods for detecting such molecular changes in lung cancer tumors are desirable. We used a nanofluidic digital PCR array platform and 16 cell lines and 20 samples of genomic DNA from resected tumors (stages I-III) to quantify the relative numbers of copies of the EGFR gene and to detect mutated EGFR alleles in lung cancer. We assessed the relative number of EGFR gene copies by calculating the ratio of the number of EGFR molecules (measured with a 6-carboxyfluorescein-labeled Scorpion assay) to the number of molecules of the single-copy gene RPP30 (ribonuclease P/MRP 30kDa subunit) (measured with a 6-carboxy-X-rhodamine-labeled TaqMan assay) in each panel. To assay for the EGFR L858R (exon 21) mutation and exon 19 in-frame deletions, we used the ARMS and Scorpion technologies in a DxS/Qiagen EGFR29 Mutation Test Kit for the digital PCR array. The digital array detected and quantified rare gefitinib/erlotinib-sensitizing EGFR mutations (0.02%-9.26% abundance) that were present in formalin-fixed, paraffin-embedded samples of early-stage resectable lung tumors without an associated increase in gene copy number. Our results also demonstrated the presence of intratumor molecular heterogeneity for the clinically relevant EGFR mutated alleles in these early-stage lung tumors. The digital PCR array platform allows characterization and quantification of oncogenes, such as EGFR, at the single-molecule level. Use of this nanofluidics platform may provide deeper insight into the specific roles of clinically relevant kinase mutations during different stages of lung tumor progression and may be useful in predicting the clinical response to EGFR-targeted inhibitors.

High‐throughput red blood cell antigen genotyping using a nanofluidic real‐time polymerase chain reaction platform

Serologic testing of donors to obtain antigen-negative blood for transfusion is limited by availability and quality of reagents. Where sequence variant information is available, molecular typing platforms can be used to determine the presence of a variant allele and offer a high-throughput format correlated to the blood group antigen. We have investigated a flexible high-throughput platform to screen blood donors for antigen genotypes in the African American population. Genomic DNA from 427 African American donors was analyzed for single-nucleotide polymorphisms responsible for red blood cell (RBC) antigens E/e, Fy(a)/Fy(b), Fy gene promoter, Jk(a)/Jk(b), Lu(a)/Lu(b), K/k, Js(a)/Js(b), Do(a)/Do(b), Jo(a), and Hy using primer/probe sets (Taqman, Applied Biosystems) on a high-throughput genotyping platform (OpenArray, BioTrove). Where available, the phenotype obtained by serologic testing was compared to genotype data. Serologic antigen types were available for 2037 of the 4270 genotypes generated. There were five discordant results. Three resolved with repeat serologic typing, one resolved after repeat genotyping, and one discordance was clarified by confirmation of the BioTrove genotype by Sanger sequencing. Triplicate determinations were made for each sample genotype and the results were identical more than 99% of the time. The nanofluidic genotyping platform described here provides an accurate method for predicting blood group phenotypes. The user-specified array layout provides flexibility of target selection and number of replicate determinations and is suitable for screening antigen types.

Biomarker Discovery and Evaluation of Response to Anti-Cancer Therapeutics in Breast Cancer Using a Novel Nanofluidic Immunoassay Platform.

Abstract Our research efforts focus on the identification and detection of fundamental molecular differences between normal and tumor cells in breast, as well as differences among distinct breast cancer subtypes, especially in terms of signal transduction pathways that control cell cycle, apoptosis and cell growth. Cancer subtype specific molecular variations dramatically affect patient responses to already existing treatments. For example, the phosphorylation status of many proteins that are involved in signal transduction pathways perturbed in cancer cells is extremely important in determining whether these cells are susceptible to killing by available cancer therapeutics. Therefore, differentially phosphorylated protein isoforms can be a particularly useful prognostic biomarker of drug response in the clinic. However, accurate detection and quantitative analysis of cancer-related phosphoproteins in tumors is limited by current technologies.Using a novel, fully automated nanocapillary electrophoresis technology (FireFlyTM) designed to separate protein molecules based on their isoelectric point (pI), we are currently developing highly sensitive assays for reliable assessment of the phosphorylation status of cancer-related phosphoproteins in tumors, before and during drug treatment. We have already developed and optimized assays measuring AKT1, AKT2, AKT3, ERK1 and ERK2, and their respective phosphoisoforms. Using these assays, we were able to measure levels of activated ERK1/2 and AKT1/2/3 in a breast cancer cell line panel developed in our lab, using protein extracted from as few as 125 cells. Based on RNA expression data, cell lines in this panel have previously been categorized in two distinct subtypes (Basal and Luminal) and their molecular phenotypes closely resemble the respective profiles of tumors obtained from breast cancer patients (CITATION). This cell line panel is extensively used to measure cellular responses to breast cancer therapeutics, including drugs that target MEK, ERK, PI3K and AKT. Using FireFly assays, we are currently measuring changes in the phosphorylation states of these targets during drug treatment, in order to completely characterize pharmacodynamic changes in these cells during treatment, and develop molecular profiles that predict response in breast cancer tumors. Since this technology enables accurate detection and quantification of protein isoforms and post-translational modifications from only very small amounts of tumor samples or serum, it promises to propel cancer biomarker discovery and enable the development of clinically useful prognostic and diagnostic assays that predict responses to drugs targeting cancer-specific molecular networks. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3172.