Nanopore blockade sensors for ultrasensitive detection of proteins in complex biological samples

Kyloon Chuah,J Justin Gooding,Yanfang Wu,Peter J Reece,Adam P Micolich,S R C Vivekchand,Katharina Gaus

doi:10.1038/s41467-019-10147-7

Abstract

Nanopore sensors detect individual species passing through a nanoscale pore. This experimental paradigm suffers from long analysis times at low analyte concentration and non-specific signals in complex media. These limit effectiveness of nanopore sensors for quantitative analysis. Here, we address these challenges using antibody-modified magnetic nanoparticles ((anti-PSA)-MNPs) that diffuse at zero magnetic field to capture the analyte, prostate-specific antigen (PSA). The (anti-PSA)-MNPs are magnetically driven to block an array of nanopores rather than translocate through the nanopore. Specificity is obtained by modifying nanopores with anti-PSA antibodies such that PSA molecules captured by (anti-PSA)-MNPs form an immunosandwich in the nanopore. Reversing the magnetic field removes (anti-PSA)-MNPs that have not captured PSA, limiting non-specific effects. The combined features allow detecting PSA in whole blood with a 0.8 fM detection limit. Our ‘magnetic nanoparticle, nanopore blockade’ concept points towards a strategy to improving nanopore biosensors for quantitative analysis of various protein and nucleic acid species.

Highlights

Nanopore sensors detect individual species passing through a nanoscale pore
We used solid-state nanopores formed in silicon nitride (SiN) with electron beam lithography (EBL) by the method shown in Supplementary Fig. 1
The blockades give robust analytical signals where large changes in current are observed as each nanopore is blocked such that small variations in pore size during fabrication, or proteins translocating through the nanopores without an associated magnetic nanoparticle do not give false signals

Summary

Introduction

Nanopore sensors detect individual species passing through a nanoscale pore This experimental paradigm suffers from long analysis times at low analyte concentration and nonspecific signals in complex media. These limit effectiveness of nanopore sensors for quantitative analysis We address these challenges using antibody-modified magnetic nanoparticles ((anti-PSA)-MNPs) that diffuse at zero magnetic field to capture the analyte, prostate-specific antigen (PSA). If the analyte is captured by the magnetic nanoparticle, the magnetic nanoparticles forms a sandwich-complex in the nanopore such that it cannot be removed when the namgnetic field is reversed to pull the nanoparticle out of the nanopore In this way false signals are avoided and better specificity is achieved. The magnetic field direction is reversed to draw any (anti-PSA)-MNPs that have not captured PSA, away from the nanopore to avoid false counts We show this approach allows lower detection limits than previously reported for nanopore sensors (sub-fM), as well as analyte specificity. The blockade design means nanopores much larger than proteins can be employed such that the nanopore blockade sensor can be used in whole blood, as shown below, without proteins that translocate through the pore giving significant resistive spikes

Methods

Results

Conclusion