Isolation and detection of single molecules on paramagnetic beads using sequential fluid flows in microfabricated polymer array assemblies

Abstract

We report a method for isolating individual paramagnetic beads in arrays of femtolitre-sized wells and detecting single enzyme-labeled proteins on these beads using sequential fluid flows in microfabricated polymer array assemblies. Arrays of femtolitre-sized wells were fabricated in cyclic olefin polymer (COP) using injection moulding based on DVD manufacturing. These arrays were bonded to a complementary fluidic structure that was also moulded in COP to create an enclosed device to allow delivery of liquids to the arrays. Enzyme-associated, paramagnetic beads suspended in aqueous solutions of enzyme substrate were delivered fluidically to the array such that one bead per well was loaded by gravity. A fluorocarbon oil was then flowed into the device to remove excess beads from the surface of the array, and to seal and isolate the femtolitre-sized wells containing beads and enzyme substrate. The device was then imaged using standard fluorescence imaging to determine which wells contained single enzyme molecules. The analytical performance of this device as the detector for digital ELISA compared favourably to the standard method, i.e., glass arrays mechanically sealed against a silicone gasket; prostate specific antigen (PSA) could be detected from 0.011 pg mL(-1) up to 100 pg mL(-1). The use of an enclosed fluidic device to isolate beads in single-molecule arrays offers a multitude of advantages for low-cost manufacturing, ease of automation, and instrument development to enable applications in biomarker validation and medical diagnosis.

High sensitivity cardiac troponin assays in the clinical laboratories

“…The CCD-camera based reader counts the number of fluorescent wells on the grid, which reflects the cTnI concentration in the specimen. Quanterix introduced the term digital ELISA to describe this type of assay [35,36]. Analytical characteristics of most ms, hs and ultrasensitive cTn assays are summarized in Table 1.…”

Rapid, Fully Automated Digital Immunoassay for p24 Protein with the Sensitivity of Nucleic Acid Amplification for Detecting Acute HIV Infection

“…After a second wash, the beads are resuspended in a resorufin ␤-Dgalactopyranoside (RGP) substrate solution. Digital processing occurs when beads are transferred to the Simoa array disc (14 ). Individual beads are then sealed in microwells in the array.…”

Transport of biomolecules to binding partners displayed on the surface of microbeads arrayed in traps in a microfluidic cell

“…In other studies, wells at the floor of the microchannel are used to sequester and array microbeads to construct the probe library. 17,18,[22][23][24] In the well geometry, target only streams over the top part of the microbead, and at high Pe thin boundary layers cannot encircle a large portion of the microbead. As a result, diffusive transport is hindered, and one solution which is proposed is to create drains in the wells to enhance convection around the microbead.…”

“…Two common methods for arraying microbeads in a microfluidic cell are by using wells patterned into the bottom surface of the cell, e.g., Duffy et al 17 and Ramsey et al 18 or traps arranged as a microfluidic obstacle course, e.g., Nehorai et al 19,20 and our prior study on arraying lipobeads. 21 Well deposition has also been enhanced by using electric and magnetic fields to assist in the capture, [22][23][24] or by using holes placed in the well and connected to a drain to provide fluid suction (see McDevitt et al 25,26 and Ketterson 27 ).…”