On-chip integration of droplet microfluidics and nanostructure-initiator mass spectrometry for enzyme screening

Lab on a Chip ◽  
2017 ◽  
Vol 17 (2) ◽  
pp. 323-331 ◽  
Author(s):  
Joshua Heinemann ◽  
Kai Deng ◽  
Steve C. C. Shih ◽  
Jian Gao ◽  
Paul D. Adams ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Droplet Microfluidics ◽  
Enzymatic Assay ◽  
Enzyme Screening ◽  
Highly Sensitive ◽  
On Chip

μNIMS, a highly sensitive and high throughput technique for enzymatic assay that integrates droplet microfluidics with nanostructure-initiator mass spectrometry (NIMS).

scholarly journals Dual sequentially addressable dielectrophoretic array for high-throughput, scalable, multiplexed droplet sorting

2021 ◽  
Vol 25 (4) ◽  
Author(s):  
Akihiro Isozaki ◽  
Dunhou Huang ◽  
Yuta Nakagawa ◽  
Keisuke Goda
Keyword(s):  
High Throughput ◽  
Cell Biology ◽  
Digital Pcr ◽  
Industrial Applications ◽  
Droplet Microfluidics ◽  
Diverse Range ◽  
Droplet Sorting ◽  
On Chip ◽  
Single Droplets

AbstractDroplet microfluidics is a powerful tool for a diverse range of biomedical and industrial applications such as single-cell biology, synthetic biology, digital PCR, biosafety monitoring, drug screening, and food, feed, and cosmetic industries. As an integral part of droplet microfluidics, on-chip multiplexed droplet sorting has recently gained enthusiasm, since it enables real-time sorting of single droplets containing cells with different phenotypes into multiple bins. However, conventional sorting methods are limited in throughput and scalability. Here, we present high-throughput, scalable, multiplexed droplet sorting by employing a pair of sequentially addressable dielectrophoretic arrays (SADAs) across a microchannel on a microfluidic chip. A SADA is an on-chip array of electrodes, each of which is sequentially activated and deactivated in synchronization to the position and speed of a flowing droplet of interest. The dual-SADA (dSADA) structure enables high-throughput deflection of droplets in multiple directions in a well-controlled manner. For proof-of-concept demonstration and characterization of the dSADA, we performed fluorescence-activated droplet sorting (FADS) with a 3-way dSADA at a high throughput of 2450 droplets/s. Furthermore, to show the scalability of the dSADA, we also performed FADS with a 5-way dSADA at a high throughput of 473 droplets/s.

scholarly journals Novel LC/MS/MS and High-Throughput Mass Spectrometric Assays for Monoacylglycerol Acyltransferase Inhibitors

2017 ◽  
Vol 22 (4) ◽  
pp. 433-439
Author(s):  
Jenson Qi ◽  
John A. Masucci ◽  
Wensheng Lang ◽  
Margery A. Connelly ◽  
Gary W. Caldwell ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Metabolic Diseases ◽  
Host Cells ◽  
Activity Assay ◽  
Highly Sensitive ◽  
Liquid Chromatography Tandem Mass ◽  
Acyltransferase Inhibitors ◽  
Tlc Analysis ◽  
Monoacylglycerol Acyltransferase

Monoacylglycerol acyltransferase enzymes (MGAT1, MGAT2, and MGAT3) convert monoacylglycerol to diacylglycerol (DAG). MGAT1 and MGAT2 are both implicated in obesity-related metabolic diseases. Conventional MGAT enzyme assays use radioactive substrates, wherein the product of the MGAT-catalyzed reaction is usually resolved by time-consuming thin layer chromatography (TLC) analysis. Furthermore, microsomal membrane preparations typically contain endogenous diacylglycerol acyltransferase (DGAT) from the host cells, and these DGAT activities can further acylate DAG to form triglyceride (TG). Our mass spectrometry (liquid chromatography–tandem mass spectrometry, or LC/MS/MS) MGAT2 assay measures human recombinant MGAT2-catalyzed formation of didecanoyl-glycerol from 1-decanoyl-rac-glycerol and decanoyl-CoA, to produce predominantly 1,3-didecanoyl-glycerol. Unlike 1,2-DAG, 1,3-didecanoyl-glycerol is proved to be not susceptible to further acylation to TG. 1,3-Didecanoyl-glycerol product can be readily solubilized and directly subjected to high-throughput mass spectrometry (HTMS) without further extraction in a 384-well format. We also have established the LC/MS/MS MGAT activity assay in the intestinal microsomes from various species. Our assay is proved to be highly sensitive, and thus it allows measurement of endogenous MGAT activity in cell lysates and tissue preparations. The implementation of the HTMS MGAT activity assay has facilitated the robust screening and evaluation of MGAT inhibitors for the treatment of metabolic diseases.

scholarly journals Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry

ACS Omega ◽  
2018 ◽  
Vol 3 (2) ◽  
pp. 1498-1508 ◽  
Author(s):  
Xue W. Diefenbach ◽  
Iman Farasat ◽  
Erik D. Guetschow ◽  
Christopher J. Welch ◽  
Robert T. Kennedy ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Protein Engineering ◽  
High Throughput ◽  
Droplet Microfluidics

Hydrophilic interaction chromatography-tandem mass spectrometry based on an amide column for the high-throughput quantification of metformin in rat plasma

RSC Advances ◽  
2015 ◽  
Vol 5 (123) ◽  
pp. 101386-101392 ◽  
Author(s):  
Rong Shi ◽  
Xining Xu ◽  
Jiasheng Wu ◽  
Tianming Wang ◽  
Yuanyuan Li ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Tandem Mass Spectrometry ◽  
High Throughput ◽  
Rat Plasma ◽  
Pharmacokinetic Studies ◽  
Tandem Mass ◽  
Sample Analysis ◽  
Hydrophilic Interaction ◽  
Chromatography Tandem Mass Spectrometry ◽  
Highly Sensitive

A simple, highly sensitive, specific, reproducible, and high-throughput Amide-HILIC-MS/MS assay to quantify metformin in rat plasma was established and successfully applied for sample analysis to support pharmacokinetic studies.

scholarly journals DNB-Based On-Chip Motif Finding (DocMF): a High-Throughput Method to Profile Different Types of Protein-DNA Interactions

2019 ◽  
Author(s):  
Zhuokun Li ◽  
Xiaojue Wang ◽  
Dongyang Xu ◽  
Dengwei Zhang ◽  
Dan Wang ◽  
...  
Keyword(s):  
Dna Binding ◽  
High Throughput ◽  
Motif Finding ◽  
High Coverage ◽  
Protospacer Adjacent Motif ◽  
Highly Sensitive ◽  
Protein Dna Interactions ◽  
High Throughput Method ◽  
On Chip ◽  
Next Generation Sequencing Ngs

ABSTRACTHere we report a highly sensitive DNB-based on-chip Motif Finding (DocMF) system that utilizes high throughput next-generation-sequencing (NGS) chips to profile protein binding or cleaving activity. Using DocMF, we successfully identified a variety of endonuclease recognition sites and the protospacer-adjacent-motif (PAM) sequences of different CRISPR systems. Our DocMF platform can simultaneously screen both 5’ and 3’ PAM regions with high coverage using the same NGS library/chip. For the well-studied SpCas9, our DocMF platform identified a small proportion of noncanonical 5’-NAG-3’ (∼5%) and 5’-NGA-3’ (∼1.6%), in addition to its common PAMs, 5’-NGG-3’ (∼89.9%). We also used the DocMF to assay two uncharacterized Cas endonucleases, VeCas9 and BvCpf1. VeCas9 PAMs were not detected by the conventional PAM depletion method. However, DocMF discovered that both VeCas9 and BvCpf1 required broader and more complicated PAM sequences for target recognition. VeCas9 preferred the R-rich motifs, whereas BvCpf1 used the T-rich PAMs. Moreover, after slightly changing the experimental protocol, we observed that dCas9, a DNA-binding protein lacking endonuclease activity, preferably binded to the previously reported PAMs 5’-NGG-3’. In summary, our studies demonstrate that DocMF is the first tool with the capacity to exhaustively assay both the binding and the cutting properties of different DNA-binding proteins.

A mesofluidic platform integrating on-chip probe ultrasonication for multiple sample pretreatment involving denaturation, reduction, and digestion in protein identification assays by mass spectrometry

The Analyst ◽  
2014 ◽  
Vol 139 (5) ◽  
pp. 992-995 ◽  
Author(s):  
J. D. Nunes-Miranda ◽  
Cristina Núñez ◽  
Hugo M. Santos ◽  
G. Vale ◽  
Miguel Reboiro-Jato ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
High Throughput ◽  
Protein Identification ◽  
Sample Pretreatment ◽  
Multiple Sample ◽  
On Chip

A novel mesofluidic platform integrating on-chip probe ultrasonication for automated high-throughput shotgun proteomic assays.

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