Digital enzyme assay using attoliter droplet array

The Analyst
Takao OnoHiroyuki Noji

Abstract

Single-molecule digital enzyme assay using micron-sized droplet array is a promising analysis method to quantify biomolecules at extremely low concentrations. However, multiplex digital enzyme assays are still difficult to access because the best buffer conditions can vary largely among enzymes. In addition, the best conditions for flurogenic compounds to retain high quantum efficiency and to avoid leakage into the oil phase can be also very different. In this study, digital enzyme assay was performed using an array of nanometer-sized droplets of 200 aL volume, termed 'nanocell'. Due to the small reaction volume, nanocell enhanced the accumulation rate of fluorescent products by a factor of 100 when compared with micron-sized reactors. Nanocell also enabled oil-free sealing of reactors: when flushed with an air flow, nanocell displayed water droplets under air, allowing enzymes to catalyze the reaction at the same rate as in oil-sealed reactors. Dual digital enzyme assay was also demonstrated using β-galactosidase and alkaline phosphatase (ALP) at pH 7.4, which is far from the optimum condition for ALP. Even under such a non-optimum condition, ALP molecules were successfully detected. Nanocell could largely expand the applicabi...Continue Reading

References

Mar 20, 1997·Nature·H NojiK Kinosita
Jun 5, 1999·Journal of Immunological Methods·C SuzukiT Nagamune
Aug 4, 1999·Proceedings of the National Academy of Sciences of the United States of America·B Vogelstein, K W Kinzler
Feb 18, 2005·Nature·Yannick RondelezHiroyuki Noji
Feb 22, 2005·Nature Biotechnology·Yannick RondelezHiroyuki Noji
Mar 14, 2007·Angewandte Chemie·Takehiko TsukaharaTakehiko Kitamori
Jul 8, 2009·Nature Nanotechnology·A K NaikM L Roukes
Oct 30, 2010·Lab on a Chip·Shouichi SakakiharaHiroyuki Noji
Jan 24, 2012·Science·Yongki ChoiPhilip G Collins
Sep 11, 2012·Lab on a Chip·Soo Hyeon KimHiroyuki Noji
Jun 4, 2014·Lab on a Chip·Daan WittersRustem F Ismagilov
Jun 18, 2014·Nature Chemical Biology·Akihiro KusumiTakahiro K Fujiwara
Jul 6, 2014·Biochimica Et Biophysica Acta·H P J Buermans, J T den Dunnen
Jul 25, 2014·Nature Communications·Rikiya WatanabeHiroyuki Noji
Oct 24, 2014·Clinical Chemistry·Jim F HuggettCarole A Foy
Sep 16, 2016·Nature·Rafael Fernandez-Leiro, Sjors H W Scheres
Jan 21, 2017·Analytical Chemistry·Yi Zhang, Hiroyuki Noji
Feb 6, 2017·Nature Protocols·Philip R NicovichKatharina Gaus
Apr 21, 2017·Biophysics Reviews·Hiroyuki NojiDuncan G G McMillan
Jan 25, 2018·The Analyst·Kentaro ShiraiTakehiko Kitamori
Feb 10, 2018·Analytical Chemistry·Barrett K DuanDavid R Walt
Mar 7, 2018·Proceedings of the National Academy of Sciences of the United States of America·Rikiya WatanabeShigekazu Nagata
May 11, 2018·The Journal of Physical Chemistry. B·Matthias J Mickert, Hans H Gorris

❮ Previous
Next ❯

Methods Mentioned

BETA
PCR
enzyme assay
enzyme-linked
ELISA
ELISAs
MDS
Fluorescence

Software Mentioned

ImageJ

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.

Related Papers

Nihon rinsho. Japanese journal of clinical medicine
Shiro Iino
Nihon rinsho. Japanese journal of clinical medicine
S Iino
© 2022 Meta ULC. All rights reserved