Evaluating the sensing performance of nanopore blockade sensors: A case study of prostate-specific antigen assay.

The detection principle of nanopore sensors relies on measuring changes in electrical signal as analyte molecules translocate through a nanoscale pore. There are two challenges with this experimental construct when using nanopores for quantitative sensing with low detection limits in complex samples. The first is getting the analyte to the nanopore in a reasonable time frame and the second is other species in the sample also translocating through the nanopore and generating erroneous signals. We have developed a nanopore blockade sensor that alleviates the limitations of diffusion-limited mass transport and non-specific signals. Antibody-modified magnetic nanoparticles are utilized to deliver analytes of interest extracted from sample to an array of antibody-modified nanopores under a controlled electromagnet, resulting in long-term nanopore blocking events due to the formation of sandwiched immunocomplexes. Herein, this study reports on understanding some of important parameters in determining the performance of nanopore blockade sensing system, where prostate-specific antigen (PSA) is used as a model analyte. We describe the characterization of nanopore blockade sensing of PSA by (1) tuning on/off the electromagnet, (2) varying nanopore number in a nanopore chip, and (3) deploying the sensor in human plasma. Results show that magnetophoresis effectively facilitates active delivery and selective sensing of PSA to the nanopore. Nanopore chips with a larger number of nanopores are shown to receive more nanopore blockades for a given concentration of analyte. Furthermore, identifiable blockade events accounted for successful detection of PSA in plasma, indicate the high specificity of the sensing system.

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

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.

The biochemiresistor: an ultrasensitive biosensor for small organic molecules.

New sensation: A resistance-based biosensor uses gold-coated magnetic nanoparticles (Au@MNPs) functionalized with the antibiotic enrofloxin (see picture; purple), which bind to anti-enrofloxin as analyte (blue). The Au@MNPs can be magnetically assembled between electrodes, and the measured resistance R is a function of analyte concentration.

Ultrasensitive electrochemical detection of prostate-specific antigen (PSA) using gold-coated magnetic nanoparticles as 'dispersible electrodes'.

Herein, we demonstrate the use of modified gold-coated magnetic nanoparticles as 'dispersible electrodes' which act as selective capture vehicles for electrochemical detection of prostate-specific antigen (PSA). A key advantage of this system is the ability to quantify non-electrochemical active analytes such as proteins with unprecedented detection limits and fast response times.

Modulation of the surface charge on polymer-stabilized gold nanoparticles by the application of an external stimulus.

A new approach to controlling the charge on gold nanoparticle surfaces is described. The method exploits the simultaneous coattachment of both charged and neutral polymers onto gold surfaces. The charged and neutral polymers were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization, and the RAFT end-group functionality was used as the anchor for attachment to gold. The approach described is general and can be applied to a wide range of monomers; those exemplified in the paper are poly(2-aminoethyl methacrylamide) (P(AEA)), poly(acrylic acid) (PAA), and poly(N,N-diemthylaminoethyl acrylate) (P(DMAEA)) together with neutral polymers based on poly(oligoethylene oxide) acrylate (P(OEG-A)). The hybrid polymer-stabilized GNPs thus formed were characterized in solution using dynamic light scattering and zeta potential measurements, transmission electron microscopy, UV-visible spectroscopy, X-ray photoelectron spectroscopy, and attenuated total reflection-Fourier-transform IR spectroscopy. The grafting densities of the polymers on GNPs were measured using thermal gravimetric analyses (TGA), as 0.4 chains/nm(2) (for PAA), 0.9 chains/nm(2) (for neutral polymers, such as P(NIPAAm), and 0.6 chain/nm(2) for the positive charged polymers P(AEA) and P(DMAEA). The directed coassembly of two different polymers (one charged and one noncharged) on the gold nanoparticle surfaces provided a mechanism (dependent on molecular weight) for shielding the surface charge imparted by the charged polymer component, allowing for a range of surface charges on the GNPs from -30 to +39 mV. In further work, the surface-charges were modulated by an external stimulus (temperature). The charge-modulation was controlled by the use of thermosensitive neutral polymers coassembled with charged polymers. The thermosensitive polymers exemplified in this paper are P(oligoethylene oxide acrylate-co-diethylene oxide acrylate) (P(OEG-A-co-DEG-A)) and P(N-isopropyl acrylamide) (P(NIPAAm). The temperature of the aqueous phase (from 15 to 70 degrees C) was then adjusted to tune the zeta potentials of the hybrid GNPs from +39 or -30 to approximately 0 mV. Finally, by manipulating the solution pH, reversible aggregation behavior of the hybrid GNPs could be induced.