Single-molecule detection of proteins with antigen-antibody interaction using resistive-pulse sensing of submicron latex particles

Abstract

We developed a resistive-pulse sensor with a solid-state pore and measured the latex agglutination of submicron particles induced by antigen-antibody interaction for single-molecule detection of proteins. We fabricated the pore based on numerical simulation to clearly distinguish between monomer and dimer latex particles. By measuring single dimers agglutinated in the single-molecule regime, we detected single human alpha-fetoprotein molecules. Adjusting the initial particle concentration improves the limit of detection (LOD) to 95 fmol/l. We established a theoretical model of the LOD by combining the reaction kinetics and the counting statistics to explain the effect of initial particle concentration on the LOD. The theoretical model shows how to improve the LOD quantitatively. The single-molecule detection studied here indicates the feasibility of implementing a highly sensitive immunoassay by a simple measurement method using resistive-pulse sensing.

Microfluidic and Nanofluidic Resistive Pulse Sensing: A Review

“…Following a similar idea, RPS detection of a cancer biomarker [ 78 ], human ferritin [ 79 ], and even single-molecule proteins [ 80 ] were also demonstrated. It should be emphasized that for RPS detection, any specific binding will be reflected by the detected electric signals.…”

Advances in High-Resolution Microscale Impedance Sensors

“…Another approach is to functionalize larger beads with target-specific receptors that cause agglomeration of beads in the presence of the target molecules. Recent examples include the use of 7 m polystyrene beads [31], 2.8 m magnetic beads [32], and 290 nm nanoparticles [33] that, combined with an 800 nm pore, allowed single-molecule resolution. Magnetic beads functionalized with specific receptors allow also the use of an external magnetic force to differentiate among cells expressing specific molecules, for instance, by using two micropores in series and slowing [34] or capturing [35] them and comparing the peaks before and after the interaction with the magnetic field.…”

A Homogeneous Multicomponent Nucleic Acid Enzyme Assay for Universal Nucleic Acid Detection by Single-Particle Inductively Coupled Plasma Mass Spectrometry

“…It can prevent these differences from being submerged in the overall signal of nanoparticles in solution, thereby improving the sensitivity of nanoparticle-based homogeneous assays. Currently developed single-particle counting techniques for homogeneous assays are mainly based on fluorescence microscopy, , dark-field microscopy, , diffraction spectroscopy, fluorescence cross correlation spectroscopy, resistive-pulse sensors, optomagnetic sensors, and inductively coupled plasma mass spectrometry (ICP-MS). − Among them, most of the optical techniques like fluorescence microscopy and dark-field microscopy rely on manual counting, which is labor-/time-intensive and imprecise, and others like resistive-pulse sensors may suffer from matrix interference. By contrast, single-particle (SP)-ICP-MS is a sensitive, accurate, automated, and matrix-resistant technique for detecting nanoparticles, in which the pulse frequency is proportional to the particle concentration of nanoparticles and the pulse intensity reflects the size of corresponding nanoparticle.…”