Subscriber access provided by University of Sussex Library
Article
Rapid Detection of Drugs in Human Plasma using a Small Molecule-Linked Hybridisation Chain Reaction Malthe Hansen-Bruhn, Line Debois Frejlev Nielsen, and Kurt Vesterager Gothelf ACS Sens., Just Accepted Manuscript • DOI: 10.1021/acssensors.8b00439 • Publication Date (Web): 14 Aug 2018 Downloaded from http://pubs.acs.org on August 15, 2018
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
ACS Sensors
Rapid Detection of Drugs in Human Plasma using a Small MoleculeLinked Hybridisation Chain Reaction Malthe Hansen-Bruhn, Line D. F. Nielsen and Kurt V. Gothelf* Center for DNA Nanotechnology (CDNA) at the Interdisciplinary Nanoscience Center (iNANO) and Department of Chemistry, Aarhus University, DK-8000 Aarhus C, Denmark. Keywords: Hybridisation Chain Reaction, Antibody, Point of Care Technology, Small Molecule Detection, Human Plasma
ABSTRACT: Rapid detection and quantification of pharmaceutical drugs directly in human plasma is of major importance for the development of relevant point of care testing devices. Here, we report a method for detection and quantification of small molecules in human plasma. An assay employing a small molecule-linked hybridization chain reaction (HCR) has been devised for the detection of the pharmaceutically relevant drugs digoxin (Dig) and methotrexate (MTX). Double modification by small molecule ligands on the initiator strand act as sites to control the rate of the HCR. Upon protein binding to the modified initiator strand, the HCR is greatly inhibited. If the protein is pre-incubated with a sample containing the small molecule analyte, the protein binding site is occupied by the analyte and the initiator strand will initiate the HCR. This enables efficient detection and quantification of small molecule analytes in nanomolar concentration even in 50% human plasma within 4 minutes. Thus, the rapidity and simplicity of this assays has potential for point of care testing.
The development of new techniques that allow fast and inexpensive detection of small molecule drugs is pertinent, since it can assist doctors in better administration of personalized doses of drugs to patients and provide the best possible treatment avoiding unwanted side-effects.1,2 The ratio between dose and blood concentration of an administered drug differs largely from patient to patient and toxic overdoses of small molecule drugs can be more efficiently avoided, if fast assessment of the plasma concentration of the compound can be made.2 One commonly employed strategy for detection of analytes is the heterogeneous assay enzyme-linked immunosorbent assay (ELISA).3 Here, assay components are immobilized and the unwanted material is removed using extensive washing steps, elongating sample turnover time.4 The golden standard in a point of care setting for small molecule detection is the very widely used glucose meter.5,6 The glucose meter combines fast assessment of analyte and convenience of use for optimal treatment, however the glucose meter is a stand-alone success in small molecule quantification in a point of care setting. In homogenous assays, the washing steps can be omitted facilitating faster sample turnover, usually with higher interference from background.4,7–9 Homogenous assays such as affinity binding assays10,11 allow for highly specific target recognition, although multivalent binding to the target is required which often is a drawback for small molecule detection. Heterogeneous assays employing protein binding induced reduction of DNA hybridisation recently resulted in de-
tection of proteins and small molecules directly in whole blood.12–14 An emerging field for homogenous small molecule detection is the use of Quenchbodies.15,16 These proteins enable rapid one-pot detection of various small molecules, but require genetic engineering of the protein. Amplification-linked homogenous assays allow for fast sample turnover and higher signal output.17 One way of achieving amplification is through enzymatic amplification of the signal. The enzymatic based amplification systems, however, often require multiple reagents, thus leading to increased costs and reduced convenience of use.18 In 2004, Pierce and co-workers19 introduced the hybridization chain reaction (HCR), which allows for nonenzymatic signal amplification in a DNA-based hybridization polymerization (Figure 1A). In this method, two complementary DNA hairpin structures coexist in solution. These so-called meta-stable hairpins are kinetically trapped. Upon addition of an initiator strand containing a toehold region (blue) and an extension that each are complementary to the toehold and stem regions on the first hairpin (HP1), HP1 is opened. The single stranded half of the HP1 contains an internal toehold (orange) that is now available for hybridization to the toehold at HP2 and in turn opens HP2. This will cause the two metastable hairpins to polymerize into a nicked double helical structure.
ACS Paragon Plus Environment
ACS Sensors 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Figure 1 The Hybridisation Chain Reaction (HCR). A) Original HCR, unmodified initiator strand enables a chain reaction, which due to co-localization of fluorophores results in a fluorescent output. B) Our work, the double modified initiator strand is able to interact with an antibody. The now sterically hindered initiator results in a retarded HCR.
A variety of designs of the HCR method have been employed for nucleic acid detection purposes.20–23 Recently, Ban et al. reported on the introduction of a small molecule label on the initiator strand, which resulted in an assay for protein detection.24 The binding of a protein to the small molecule ligand retarded the HCR due to the change in sterical properties of the initiator strand. This technique allowed for the efficient detection of proteins (streptavidin, folate receptor) over the course of 3 hours using the pyrene excimer effect as a reporter. According to Huang et al.25, the pyrene excimer effect is incompatible with detection in biological fluids, unless sophisticated fluorescence methods are employed. A common drawback of the HCR is the slow propagation speed of the system. However, in a recent study by Ang et al.26, optimization of the meta-stable hairpin design rules resulted in faster propagation (
