There is increasing interest in using nanopores in synthetic membranes as resistive-pulse sensors for molecular and macromolecule analytes. In general, this method entails measuring current pulses associated with translocation of the analyte through the nanopore sensor element. A key challenge for this sensing paradigm is building selectivity into the protocol so that the current pulses for the target analyte can be distinguished from current pulses for other species that might be present in the sample. We show here that this can be accomplished with a protein analyte by adding to the solution an antibody that selectively binds the protein. We demonstrate this concept using bovine serum albumin (BSA) and a Fab fragment from a BSA-binding polyclonal antibody. Because the complex formed upon binding of the Fab to BSA is larger than the free BSA molecule, the current-pulse signature for the BSA/Fab complex can be easily distinguished from the free BSA. Furthermore, the BSA/Fab pulses can be easily distinguished from the pulses obtained for the free Fab and from pulses obtained for a control protein that does not bind to the Fab. Finally, we also show that the current-pulse signature for the BSA/Fab complex can provide information about the size and stoichiometry of the complex.

We demonstrate here a new electrokinetic phenomenon, Electroosmotic flow (EOF) rectification, in synthetic membranes containing asymmetric pores. Mica membranes with pyramidally shaped pores prepared by the track-etch method were used. EOF was driven through these membranes by using an electrode in solutions on either side to pass a constant ionic current through the pores. The velocity of EOF depends on the polarity of the current. A high EOF velocity is obtained when the polarity is such that EOF is driven from the larger base opening to the smaller tip opening of the pore. A smaller EOF velocity is obtained when the polarity is reversed such that EOF goes from tip to base. We show that this rectified EOF phenomenon is the result of ion current-rectification observed in such asymmetric-pore membranes.

A consumer-grade fused filament fabrication (FFF) 3D printer was used to construct fluidic devices for nanoparticle preparation and electrochemical sensing. Devices were printed using poly(ethylene terephthalate) and featured threaded ports to connect polyetheretherketone (PEEK) tubing via printed fittings prepared from acrylonitrile butadiene styrene (ABS). These devices included channels designed to have 800 × 800 µm2 square cross sections and were semitransparent to allow visualization of the solution-filled channels. A 3D-printed device with a Y-shaped mixing channel was used to prepare Prussian blue nanoparticles (PBNPs) under flow rates of 100 to 2000 µL min−1. PBNPs were then attached to gold electrodes for hydrogen peroxide sensing. 3D-printed devices used for electrochemical measurements featured threaded access ports into which a fitting equipped with reference, counter, and PBNP-modified working electrodes could be inserted. PBNP-modified electrodes enabled amperometric detection of H2O2 in the 3D-printed channel by flow-injection analysis, exhibiting a detection limit of 100 nM and linear response up to 20 µM. These experiments show that a consumer-grade FFF printer can be used to fabricate low-cost fluidic devices for applications similar to those that have been reported with more expensive 3D-printing methods.

Nanomaterials and biomaterials are important components of new electrochemical arrays designed for sensitive detection of proteins in biological fluids. Such multiplexed protein arrays are predicted to have an important future in personalized medical diagnostics, especially for cancer and heart disease. Sandwich immunoassays for proteins benefit greatly in sensitivity from the use of nanostructured sensor surfaces and multilabeled detection strategies involving nano- or microparticles. In these assays, capture agents such as antibodies or aptamers are attached to sensor surfaces in the array. Target proteins with large binding constants for the affinity agents are captured from liquid samples with high efficiency, either on the sensors or on magnetic bioconjugate particles decorated with many copies of labels and antibodies. After target proteins are captured on the sensor surfaces, the labels are detected by electrochemical techniques. This feature article begins with an overview of the recent history of nanoparticles in electrochemical protein sensors, then moves on to specific examples from our own laboratories. We discuss fabrication of nanostructured sensors and arrays with the aim of multiplexed detection as well as reusability. Following this, we describe systems that integrate particle-based protein sensing with microfluidics for multiplexed protein detection. We end with predictions on the diagnostic future of protein detection.