Low-abundance Targets Research Articles

Sensitive and specific detection of tumour cells based on a multivalent DNA nanocreeper and a multiplexed fluorescence supersandwich.

A self-assembled DNA nanostructure based on a DNA nanocreeper and multiplexed fluorescence supersandwich was designed for the sensitive and specific detection of tumour cells. This nanostructure could improve the binding affinity of current aptamers and trigger signal amplification, which provide potential for the discrimination of low abundant target cells in liquid biopsy.

A fluorescent nanosphere-based immunochromatography test strip for ultrasensitive and point-of-care detection of tetanus antibody in human serum.

Tetanus still possesses a high infection risk and leads a number of human deaths in poor nations. Point-of-care and ultrasensitive detection of tetanus antibody levels in serum is the key to decrease the risk of tetanus infection and improve the health of people. In this work, by using ultra bright fluorescent nanospheres (FNs) and portable lateral flow test strip (LFTS), a point-of-care and ultrasensitive sensing method has been developed for the detection of tetanus antibodies in human serum. This assay works quite well for tetanus antibodies in the concentration range from 0.0002 to 0.0220IU/mL with a low detection limit of 0.00011IU/mL, which is 100-fold lower than conventional gold-based LFTSs. The high sensitivity makes this method suitable for use to detect the low-abundant target in real samples. Besides, this cost-effective FN-based LFTS assay possesses good selectivity, high accuracy, and satisfactory reliability, which holds great potential as a robust candidate for routine medical diagnosis and rapid home testing. Graphical abstract.

Catalytic Self-Assembly of Quantum-Dot-Based MicroRNA Nanosensor Directed by Toehold-Mediated Strand Displacement Cascade.

Semiconductor quantum dots (QDs) are highly attractive nanomaterials with wide biomedical applications owing to their unique photophysical properties. However, the adaptation of the nonenzymatic QD nanosensor assembly to sense low-abundance targets remains a great challenge. Herein, taking advantage of the dynamic DNA nanotechnology and single-molecule fluorescence detection, we demonstrate the catalytic self-assembly of a QD-based microRNA nanosensor directed by toehold-mediated strand displacement cascade for the simple and sensitive detection of microRNA at femtomolar concentration without the requirement of any enzymes. Moreover, this QD nanosensor is capable of detecting circulating microRNA in clinical serum samples and even imaging microRNA in living cells. This work may extend the use of an enzyme-free QD nanosensor assembly for low-abundance biomarker detection and offer a novel platform for fundamental biomedical research and clinical applications.

Simply Mixing Poly Protein G with Detection Antibodies Enhances the Detection Limit and Sensitivity of Immunoassays.

An insufficient amount of detection antibodies bound to their antigens usually limits the sensitivity of immunoassays. Here, we describe a simple method to improve the detection limit and sensitivity of various immunoassays by mixing detection antibodies with a soluble poly protein G (named 8pG). 8pG was developed by fusing eight repeated fragment crystallizable (Fc) binding domains of streptococcal protein G to a linear polymer. Simply mixing detection antibodies with 8pG to form an antibody/8pG complex largely increased the accumulation of detection antibody to target molecules, which dramatically enhanced the sensitivity in direct ELISA, sandwich ELISA, Western blot, and flow cytometry systems, separately. The detection limit of Western blot for low-abundance PEGylated interferon (Pegasys) and recombinant human CTLA4 (rhCTLA4) improved by at least 13-fold and 31-fold, respectively, upon mixing detection antibodies with 8pG. Moreover, the nanoscale size of the antibody/8pG complex did not influence the granularity and dimension of target cells in the flow cytometry system. Collectively, we provide a quick and easy-to-operate method to make various immunoassays to sensitively detect low-abundance target molecules by just mixing their detection antibodies with 8pG.

Breaking Through Bead-Supported Assay: Integration of Optical Tweezers Assisted Fluorescence Imaging and Luminescence Confined Upconversion Nanoparticles Triggered Luminescent Resonance Energy Transfer (LRET).

Herein, a conceptual approach for significantly enhancing a bead-supported assay is proposed. For the fluorescence imaging technology, optical tweezers are introduced to overcome the fluid viscosity interference and immobilize a single tested bead at the laser focus to guarantee a fairly precise imaging condition. For the selection of fluorescent materials and the signal acquisition means, a type of innovative luminescence confined upconversion nanoparticle with a unique sandwich structure is specially designed to act as an efficient energy donor to trigger the luminescent resonance energy transfer (LRET) process. By further combining the double breakthrough with a molecular beacon model, the newly developed detection strategy allows for achieving a pretty high LRET ratio (≈ 88%) to FAM molecules and offering sound assay performance toward miRNA analysis with a detection limit as low as the sub-fM level, and is capable of well identifying single-base mismatching. Besides, this approach not only is able to accurately qualify the low-abundance targets from as few as 30 cancer cells but also can be employed as a valid cancer early warning tool for performing liquid biopsy.

Detection of Low-Abundance Metabolites in Live Cells Using an RNA Integrator

Genetically encoded biosensors are useful tools for detecting the presence and levels of diverse biomolecules in living cells. However, low-abundance targets are difficult to detect because they are often unable to bind and activate enough biosensors to detect using standard microscopic imaging approaches. Here we describe a type of RNA-based biosensor, an RNA integrator, which enables detection of low-abundance targets invitro and in living cells. The RNA integrator is an RNA sequence comprising a ribozyme and an unfolded form of thefluorogenic aptamer Broccoli. Upon binding itstarget, the ribozyme undergoes cleavage and releases Broccoli, which subsequently folds and becomes fluorescent. Importantly, each target molecule can bind and induce cleavage of multiple copies of the integrator sensor, resulting in an amplified signal. We show that this approach can be generalized to numerous different ribozyme types for the detection of various small molecules.

An efficient and cost-effective method for purification of small sized DNAs and RNAs from human urine.

Urine holds great promise as a non-invasive sampling method for molecular diagnostics. The cell-free nucleic acids of urine however are small, labile, and difficult to purify. Here an efficient method for the purification of these nucleic acids is presented. An empirically derived protocol was devised by first identifying conditions that allowed recovery of a 100 base pair (bp) DNA, followed by optimization using a quantitative polymerase chain reaction (qPCR) assay. The resulting method efficiently purifies both small sized DNAs and RNAs from urine, which when combined with quantitative reverse transcription PCR (qRTPCR), demonstrably improves detection sensitivity. Fractionation experiments reveal that nucleic acids in urine exist both in the cell-free and cellular fraction, roughly in equal proportion. Consistent with previous studies, amplicons > 180bp show a marked loss in PCR sensitivity for cell-free nucleic acids. Finally, the lysis buffer developed here also doubles as an effective preservative, protecting against nucleic acid degradation for at least two weeks under simulated field conditions. With this method, volumes of up to 25ml of whole urine can be purified in a high-throughput and cost-effective manner. Coupled with its ability to purify both DNA and RNA, the described method may have broad applicability for improving the diagnostic utility of urine, particularly for the detection of low abundant targets.

A Targeted LC-MS Strategy for Low-Abundant HLA Class-I-Presented Peptide Detection Identifies Novel Human Papillomavirus T-Cell Epitopes.

For rational design of therapeutic vaccines, detailed knowledge about target epitopes that are endogenously processed and truly presented on infected or transformed cells is essential. Many potential target epitopes (viral or mutation‐derived), are presented at low abundance. Therefore, direct detection of these peptides remains a challenge. This study presents a method for the isolation and LC‐MS3‐based targeted detection of low‐abundant human leukocyte antigen (HLA) class‐I‐presented peptides from transformed cells. Human papillomavirus (HPV) was used as a model system, as the HPV oncoproteins E6 and E7 are attractive therapeutic vaccination targets and expressed in all transformed cells, but present at low abundance due to viral immune evasion mechanisms. The presented approach included preselection of target antigen‐derived peptides by in silico predictions and in vitro binding assays. The peptide purification process was tailored to minimize contaminants after immunoprecipitation of HLA‐peptide complexes, while keeping high isolation yields of low‐abundant target peptides. The subsequent targeted LC‐MS3 detection allowed for increased sensitivity, which resulted in successful detection of the known HLA‐A2‐restricted epitope E711–19 and ten additional E7‐derived peptides on the surface of HPV16‐transformed cells. T‐cell reactivity was shown for all the 11 detected peptides in ELISpot assays, which shows that detection by our approach has high predictive value for immunogenicity. The presented strategy is suitable for validating even low‐abundant candidate epitopes to be true immunotherapy targets.

Culture-Free Detection of Bacterial Pathogens on Plasmonic Nanopillar Arrays Using Rapid Raman Mapping.

We utilized a fast Raman spectral mapping technique for fast detection of bacterial pathogens. Three-dimensional (3D) plasmonic nanopillar arrays were fabricated using the nanolithography-free process consisting of maskless Ar plasma treatment of a polyethylene terephthalate substrate and subsequent metal deposition. Bacterial pathogens were immobilized on the positively charged poly(l-lysine)-coated 3D plasmonic substrate through electrostatic interactions. Then, the bacterial surfaces were selectively labeled with antibody-conjugated surface-enhanced Raman scattering (SERS) nanotags, and Raman mapping images were collected and statistically analyzed for quantitative analysis of bacteria. Salmonella typhimurium was selected as a model pathogen bacterium to confirm the efficacy of our SERS imaging technique. Minimum number of Raman mapping points with statistical reliability was determined to reduce assay time. It was possible to get a statistically reliable standard calibration curve for 529 pixels (laser spot with 60 μm interval), which required a total mapping time of 45 min to get a standard calibration curve for five different concentrations of bacteria in the 0 to 106 CFU/mL range. No amplification step was necessary for quantification because low-abundance target bacteria could be measured using the Raman spectral mapping technique. Therefore, this approach allows accurate quantification of bacterial pathogens without any culturing or enrichment process.

Droplet Digital PCR for Quantitation of Extracellular RNA.

Cell-to-cell communication involves the release of biological molecules into the extracellular space and their uptake by recipient cells. These molecules include RNA thatcan modulate cellular signaling and biological processes. To study extracellular RNA, highly sensitive and precise methodsfor their detection are needed. Digital polymerase chain reaction (dPCR) can be a useful method for detecting and analyzing extracellular RNA. The sensitivity of digital PCR can exceed that of quantitative PCR for low abundance targets such as extracellular RNA.

Quantification of Circulating MicroRNAs by Droplet Digital PCR.

MicroRNAs (miRNAs) are released in the blood as cell-free molecules either linked to Ago proteins and LDL or enveloped inside exosomes and microvescicles. The amount of specific circulating microRNAs has been discovered to change accordingly to a disease state and to be potentially used as a disease biomarker. Sensitive and accurate methods for circulating microRNA quantification using probe-based or dye-based digital PCR technology have been developed. With a digital PCR system it is possible to obtain the absolute quantification of specific miRNAs, bypassing several issues related to low abundance targets and miRNA normalization. This chapter addresses the workflow and methods for miRNA assessment in biological fluids using EvaGreen-based droplet digital PCR as well as how to analyze and interpret results.

Abstract 217: Quantitative analysis of two cancer signaling pathways using multiplex immunoprecipitation and targeted mass spectrometry

Abstract Background: The AKT/mTOR and RAS/ERK pathways represent key mechanisms for cells to regulate cell survival, proliferation, and motility. In addition to their independent signaling cascades, which provide offsetting mechanisms, these two pathways extensively engage in cross-talk in order to both positively and negatively regulate each other. The quantitation of protein expression and modifications of pathway targets are critical for characterization of disease, monitoring cancer progression and determining treatment response. Some major bottlenecks in the accurate quantitation of these proteins are the lack of rigorously validated reagents and a reliance on semi-quantitative results from available immunoassays. Mass spectrometry (MS) is increasingly becoming the detection method of choice for proteins and their post-translational modifications (PTMs). Immunoprecipitation coupled with MS (IP-MS) enables assessment of antibody specificity and identification of low-abundant targets. Multiplexed IP coupled with targeted MS (mIP-tMS) can quantitate multiple proteins of interest, PTMs and interacting partners in a single MS run. The objective of this study was to determine the efficacy of mIP-tMS for quantitation of the AKT/mTOR and RAS/ERK pathway proteins and to compare these results versus Western blotting (WB). Methods: Serum starved and LY294002 treated HCT116 and A549 cells were stimulated with IGF-1. Antibodies to targets in the AKT/mTOR and RAS/ERK pathways were selected for verification of antibody specificity by IP-MS. mIP-tMS assays were developed and validated for absolute quantitation of targets in these pathways and benchmarked with WB across two unstimulated, IGF-1 stimulated and LY294002 treated cell lysates as well as several tissue lysates. Results: Previously, we showed that an optimized IP-MS workflow for Protein A/G and Streptavidin magnetic beads increases target protein yield with low non-specific background. In this study, we validated antibodies to several AKT/mTOR and RAS/ERK pathway targets using the optimized IP-MS workflow. mIP-tMS assays allowed absolute quantitation for multiple total and phosphorylated targets from both pathways in low to sub-nanogram concentrations across two unstimulated, IGF-1 stimulated and LY294002 treated cell lysates. The benchmarking of mIP-tMS assays showed low correlation for quantitation of total and phosphorylated targets relative to WB. This lower correlation may be due to differences in the specificity of antibodies used for each assay technique. Conclusion: Overall, the mIP-tMS assays can be used for effective identification and quantitation of AKT/mTOR and RAS/ERK pathway proteins in multiple cancer cell lines and tissue samples. Major advantages of this assay are high confidence in target identity coupled with simultaneous absolute quantitation of multiple targets and their PTMs from cancer signaling pathways. Note: This abstract was not presented at the meeting. Citation Format: Bhavin Patel, Leigh Foster, Gregory Potts, Abid Haseeb, Alex Behling, John Rogers. Quantitative analysis of two cancer signaling pathways using multiplex immunoprecipitation and targeted mass spectrometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 217. doi:10.1158/1538-7445.AM2017-217

Droplet Digital PCR versus qPCR for gene expression analysis with low abundant targets: from variable nonsense to publication quality data

Quantitative PCR (qPCR) has become the gold standard technique to measure cDNA and gDNA levels but the resulting data can be highly variable, artifactual and non-reproducible without appropriate verification and validation of both samples and primers. The root cause of poor quality data is typically associated with inadequate dilution of residual protein and chemical contaminants that variably inhibit Taq polymerase and primer annealing. The most susceptible, frustrating and often most interesting samples are those containing low abundant targets with small expression differences of 2-fold or lower. Here, Droplet Digital PCR (ddPCR) and qPCR platforms were directly compared for gene expression analysis using low amounts of purified, synthetic DNA in well characterized samples under identical reaction conditions. We conclude that for sample/target combinations with low levels of nucleic acids (Cq ≥ 29) and/or variable amounts of chemical and protein contaminants, ddPCR technology will produce more precise, reproducible and statistically significant results required for publication quality data. A stepwise methodology is also described to choose between these complimentary technologies to obtain the best results for any experiment.

Synthesis of a Highly Azide-Reactive and Thermosensitive Biofunctional Reagent for Efficient Enrichment and Large-Scale Identification of O-GlcNAc Proteins by Mass Spectrometry.

O-linked β-N-acetylglucosamine (O-GlcNAc) is a ubiquitous post-translational modification of proteins in eukaryotic cells. Despite their low abundance, O-GlcNAc-modified proteins play many important roles in regulating gene expression, signal transduction, and cell cycle. Aberrant O-GlcNAc proteins are correlated with many major human diseases, such as Alzheimer's disease, diabetes, and cancer. Because of the extremely low stoichiometry of O-GlcNAc proteins, enrichment is required before mass spectrometry analysis for large-scale identification and in-depth understanding of their cellular function. In this work, we designed and synthesized a novel thermosensitive immobilized triarylphosphine reagent as a convenient tool for efficient enrichment of azide-labeled O-GlcNAc proteins from complex biological samples. Immobilization of triarylphosphine on highly water-soluble thermosensitive polymer largely increases its solubility and reactivity in aqueous solution. As a result, facilitated coupling is achieved between triarylphosphine and azide-labeled O-GlcNAc proteins via Staudinger ligation, due to the increased triarylphosphine concentration, reduced interfacial mass transfer resistance, and steric hindrance in homogeneous reaction. Furthermore, solubility of the polymer from complete dissolution to full precipitation can be easily controlled by simply adjusting the environmental temperature. Therefore, facile sample recovery can be achieved by increasing the temperature to precipitate the polymer-O-GlcNAc protein conjugates from solution. This novel immobilized triarylphosphine reagent enables efficient enrichment and sensitive detection of more than 1700 potential O-GlcNAc proteins from HeLa cell using mass spectrometry, demonstrating its potential as a general strategy for low-abundance target enrichment.

Toehold Mediated One-Step Conformation-Switchable “Signal-On” Electrochemical DNA Sensing Enhanced with Homogeneous Enzymatic Amplification

The development of highly sensitive and sequence-specific electrochemical DNA (E-DNA) sensors, featuring flexible, one-step, and "signal-on" design, is a long-lasting goal. Here, we present a single-step, toehold-triggered structure-switchable signaling design that is "signal-on" and compatible with homogeneous enzyme-assisted target recycling (EATR). In this design, a partially hybridized duplex is bifunctional, which consists of a signal probe having foldable hairpin sequence and a target recognition probe with exposed toehold domain. In the presence of both target and exonuclease, the toehold sequence rapidly fuels the strand displacement reaction, liberating the surface-confined toehold-target duplex into homogeneous solution for target recycling and meanwhile leaving the dehybridized signal probe to form a stem-loop structure for signaling. Through such an 1:N enzymatic catalysis, more and more unfolded probes self-hybridize to their original folded configuration, giving a remarkable signal gain. This enzyme-assisted toehold E-DNA (etE-DNA) sensor achieves a satisfactory detection limit down to 42 fM, which is lower than that of the routine switchable E-DNA sensor by several orders of magnitude. In addition, the strategy shows high selectivity against a single-base mismatch and is capable of probing low abundant target DNA directly in human serum with minimal interference. By synergizing the toehold-based high selectivity, EATR, and one-step conformation-switchable signaling, this functional etE-DNA sensor appears to be a promising bioassay approach for clinical diagnostics.

T Lymphocyte Immunophenotyping: 14-Color Flow Cytometry Panel Design Using the Attune NxT Flow Cytometer and Super Bright Fluorescent Dyes

Abstract The Invitrogen™ eBioscience™ Super Bright polymer dyes represent a suite of bright fluorophores excited by the violet laser (405 nm). Optimized for use in flow cytometry, the Super Bright dyes allow for expanded use of violet laser excitation and promote streamlined multicolor panel design. Due to their inherent brightness, detection of cell populations with low abundance targets is possible. A 14-color panel used to characterize human T-cell lymphocytes is presented, with information on differentiation profiles, activation or exhaustion status, and co-stimulation activity. Both cryopreserved human peripheral blood mononuclear cells (PBMC) and fresh blood collected from a normal donor was used. Acquisition and analysis was performed using the Attune™ NxT Flow Cytometer four laser system, which has the ability to detect multiple violet-excited fluorophores. The results show well characterized cell populations, with expected changes between unstimulated and stimulated PBMC ex vivo. Correction for spectral overlap was performed using standard instrument auto-compensation procedures. The unique acoustic focusing technology of Attune NxT Flow Cytometer enables rapid and accurate detection with sensitivity at high flow rates without compromise of data quality, as demonstrated with this 14-color data set. The Attune NxT cytometer is designed to accommodate a wide variety of experimental conditions and combined with the Super Bright dyes provides expanded choice for panel design. For research use only. Not for use in diagnostic procedures.

Poly-protein G-expressing bacteria enhance the sensitivity of immunoassays

The sensitivities of solid-phase immunoassays are limited by the quantity of detection antibodies bound to their antigens on the solid phase. Here, we developed a poly-protein G-expressing bacterium as an antibody-trapping microparticle to enhance the signals of immunoassays by increasing the accumulation of detection antibodies on the given antigen. Eight tandemly repeated fragment crystallisable (Fc) binding domains of protein G were stably expressed on the surface of Escherichia coli BL21 cells (termed BL21/8G). BL21/8G cells showed a higher avidity for trapping antibodies on their surface than monomeric protein G-expressing BL21 (BL21/1G) cells did. In the sandwich enzyme-linked immunosorbent assay (ELISA), simply mixing the detection antibody with BL21/8G provided a detection limit of 6 pg/mL for human interferon-α (IFN-α) and a limit of 30 pg/mL for polyethylene glycol (PEG)-conjugated IFN-α (Pegasys), which are better than that of the traditional ELISA (30 pg/mL for IFN-α and 100 pg/mL for Pegasys). Moreover, the sensitivity of the Western blot for low-abundance Pegasys (0.4 ng/well) was increased by 25 folds upon mixing of an anti-PEG antibody with BL21/8G cells. By simply being mixed with a detection antibody, the poly-protein G-expressing bacteria can provide a new method to sensitively detect low-abundance target molecules in solid-phase immunoassays.

Comprehensive and quantitative mapping of RNA–protein interactions across a transcribed eukaryotic genome

RNA-binding proteins (RBPs) control the fate of nearly every transcript in a cell. However, no existing approach for studying these posttranscriptional gene regulators combines transcriptome-wide throughput and biophysical precision. Here, we describe an assay that accomplishes this. Using commonly available hardware, we built a customizable, open-source platform that leverages the inherent throughput of Illumina technology for direct biophysical measurements. We used the platform to quantitatively measure the binding affinity of the prototypical RBP Vts1 for every transcript in the Saccharomyces cerevisiae genome. The scale and precision of these measurements revealed many previously unknown features of this well-studied RBP. Our transcribed genome array (TGA) assayed both rare and abundant transcripts with equivalent proficiency, revealing hundreds of low-abundance targets missed by previous approaches. These targets regulated diverse biological processes including nutrient sensing and the DNA damage response, and implicated Vts1 in de novo gene "birth." TGA provided single-nucleotide resolution for each binding site and delineated a highly specific sequence and structure motif for Vts1 binding. Changes in transcript levels in vts1Δ cells established the regulatory function of these binding sites. The impact of Vts1 on transcript abundance was largely independent of where it bound within an mRNA, challenging prevailing assumptions about how this RBP drives RNA degradation. TGA thus enables a quantitative description of the relationship between variant RNA structures, affinity, and in vivo phenotype on a transcriptome-wide scale. We anticipate that TGA will provide similarly comprehensive and quantitative insights into the function of virtually any RBP.

Abstract 3884: Quantitative analysis of IGF1R/AKT/mTOR pathway using multiplex immunoprecipitation and targeted mass spectrometry

Abstract Background: The quantitative measurement of protein expression and modification status of AKT/mTOR pathway proteins is necessary for precise characterization of the disease, monitoring cancer progression and determining treatment response. A major bottleneck in the quantitation of signaling pathway proteins is the lack of rigorously validated methods/reagents and a reliance on semi-quantitative results from current immunoassay technologies (Western blot, ELISA and Luminex). Mass Spectrometry (MS) is increasingly becoming the detection methodology of choice for proteins and their post-translational modifications (PTMs). Immunoprecipitation (IP) is commonly used upstream of MS as an enrichment tool for low-abundant protein targets. In addition to protein identification, IP can be combined with targeted MS to quantitate proteins of interest and identify protein-protein interactions. The objective of this study was to determine the efficacy of multiplex IP to targeted MS technique for measurement of the total and phosphorylated AKT/mTOR pathway targets and to evaluate whether multiplex IP-MS assays are as effective as the current single-plex immunoassay (WB and ELISA) and multiplex Luminex assays. Methods: Serum starved HCT116, MCF7 and A549 cells were stimulated with IGF-1. Multiplex IP to targeted MS assays (mIP-tMS) were developed and validated for absolute quantitation of eleven total and ten phosphorylated AKT/mTOR pathway targets (IGF1R, IR, IRS1, PTEN, AKT, mTOR, GSK3α, GSK3β, TSC2, p70S6K and PRAS40). Validated mIP-tMS assays were benchmarked against currently available WB, ELISA and multiplex Luminex immunoassays across three unstimulated and IGF-1 stimulated cell lysates. Results: In previous work, we showed that an optimized IP-MS workflow for Protein A/G and Streptavidin magnetic beads can increase target protein yield with low non-specific background. In this study, we validated multiple antibodies for eleven total and ten phosphorylated AKT/mTOR pathway targets using the optimized IP-MS workflow. mIP-tMS assays allowed absolute quantitation for all eleven total and ten phosphorylated targets in low to sub nanogram concentrations across three unstimulated and IGF-1 stimulated cell lysates. The benchmarking of mIP-tMS assays showed high correlation for quantitation of total target relative abundance compared to WB, ELISA and Luminex assays. However, for some phosphorylated targets, mIP-tMS assays had low concordance to the other immunoassays possibly due to differences in the specificity of anti-phospho antibodies used for each assay. Conclusion: Overall, the multiplex targeted MS assay can be used for identification and quantification of AKT/mTOR pathway proteins in cancer cell lines or tissue samples. Major advantages of this mIP-tMS assay are high confidence in target identity coupled with simultaneous quantitation of multiple targets and their PTMs. Citation Format: Bhavin Patel, Alex Behling, Leigh Foster, Ryan Bomgarden, Carrie Clothier, Kay Opperman, John Rogers. Quantitative analysis of IGF1R/AKT/mTOR pathway using multiplex immunoprecipitation and targeted mass spectrometry. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3884.

A Microfiltration Device for Urogenital Schistosomiasis Diagnostics.

Schistosomiasis is a parasitic disease affecting over 200 million people worldwide. This study reports the design and development of a microfiltration device for isolating schistosome eggs in urine for rapid diagnostics of urogenital schistosomiasis. The design of the device comprises a linear array of microfluidic traps to immobilize and separate schistosome eggs. Sequential loading of individual eggs is achieved autonomously by flow resistance, which facilitates observation and enumeration of samples with low-abundance targets. Computational fluid dynamics modeling and experimental characterization are performed to optimize the trapping performance. By optimizing the capture strategy, the trapping efficiency could be achieved at 100% with 300 μl/min and 83% with 3000 μl/min, and the filtration procedure could be finished within 10 min. The trapped eggs can be either recovered for downstream analysis or preserved in situ for whole-mount staining. On-chip phenotyping using confocal laser fluorescence microscopy identifies the microstructure of the trapped schistosome eggs. The device provides a novel microfluidic approach for trapping, counting and on-chip fluorescence characterization of urinal Schistosoma haematobium eggs for clinical and investigative application.