Label-Free, Non-Optical Readout of Bead-Based Immunoassays with and without Electrokinetic Preconcentration.

In this article, we report a microfluidic bead-based lateral flow immunoassay (LFIA) with a novel sensing mechanism for label-free, non-optical detection of protein binding. This device comprises two packed beds of microbeads: first, bioconjugated microbeads that serve as a test line, and second, a three-dimensional (3D) electrode for sensing. As the protein target binds the bioconjugated microbeads, a shift in ionic conductivity across the bioconjugated beads is produced and can be directly measured at the surface of the 3D electrode by obtaining current-voltage curves before and after incubation of the analyte. We use a model antigen, rabbit IgG, for quantitative evaluation of this sensor, obtaining a limit of detection (LOD) of 50 nM for the LFIA. We demonstrate that this device can be used to measure binding kinetics, exhibiting a rapid (<3 min) increase in the signal after the introduction of the analyte and an exponential decay in the signal after replacing the sample with buffer only. To improve the LOD of our system, we implement an electrokinetic preconcentration technique, faradaic ion concentration polarization (fICP), to increase the local concentration of antigen available during binding as well as the time the antigen interacts with the test line. Our results indicate that this enrichment-enhanced assay (fICP-LFIA) has an LOD of 370 pM, an 135-fold improvement over the LFIA and a 7-fold improvement in sensitivity. We anticipate that this device can be readily adapted for point-of-care diagnostics and translated to any desired protein target by simply modifying the biorecognition agent on these off-the-shelf microbeads.

Electrokinetic Enrichment and Label-Free Electrochemical Detection of Nucleic Acids by Conduction of Ions along the Surface of Bioconjugated Beads.

In this paper, we report a method to integrate the electrokinetic pre-enrichment of nucleic acids within a bed of probe-modified microbeads with their label-free electrochemical detection. In this detection scheme, hybridization of locally enriched target nucleic acids to the beads modulates the conduction of ions along the bead surfaces. This is a fundamental advancement in that this mechanism is similar to that observed in nanopore sensors, yet occurs in a bed of microbeads with microscale interstices. In application, this approach has several distinct advantages. First, electrokinetic enrichment requires only a simple DC power supply, and in combination with nonoptical detection, it makes this method amenable to point-of-care applications. Second, the sensor is easy to fabricate and comprises a packed bed of commercially available microbeads, which can be readily modified with a wide range of probe types, thereby making this a versatile platform. Finally, the sensor is highly sensitive (picomolar) despite the modest 100-fold pre-enrichment we employ here by faradaic ion concentration polarization (fICP). Further gains are anticipated under conditions for fICP focusing that are known to yield higher enrichment factors (up to 100,000-fold enrichment). Here, we demonstrate the detection of 3.7 pM single-stranded DNA complementary to the bead-bound oligoprobe, following a 30 min single step of enrichment and hybridization. Our results indicate that a shift in the slope of a current-voltage curve occurs upon hybridization and that this shift is proportional to the logarithm of the concentration of target DNA. Finally, we investigate the proposed mechanism of sensing by developing a numerical simulation that shows an increase in ion flux through the bed of insulating beads, given the changes in surface charge and zeta potential, consistent with our experimental conditions.