Ultrafast immunoassays by coupling dielectrophoretic biomarker enrichment in nanoslit channel with electrochemical detection on graphene

Abstract

Heterogeneous immunoassays usually require long incubation times to promote specific target binding and several wash steps to eliminate non-specific binding. Hence, signal saturation is rarely achieved at detection limit levels of analyte, leading to significant errors in analyte quantification due to extreme sensitivity of the signals to incubation time and methodology. The poor binding kinetics of immunoassays at detection limit levels can be alleviated through creating an enriched analyte plug in the vicinity of immobilized capture probes to enable signal saturation at higher levels and at earlier times, due to higher analyte association and its faster replenishment at the binding surface. Herein, we achieve this by coupling frequency-selective dielectrophoretic molecular dam enrichment of the target biomarker in physiological media to capture probes immobilized on graphene-modified surfaces in a nanoslit to enable ultrafast immunoassays with near-instantaneous (<2 minutes) signal saturation at dilute biomarker levels (picomolar) within ultra-low sample volumes (picoliters). This methodology is applied to the detection of Prostate Specific Antigen (PSA) diluted in serum samples, followed by validation against a standard two-step immunoassay using three de-identified patient samples. Based on the ability of dielectrophoretic molecular dam analyte enrichment methods to enable the detection of PSA at 1-5 pg mL(-1) levels within a minute, and the relative insensitivity of the signals to incubation time after the first two minutes, we envision its application for improving the sensitivity of immunoassays and their accuracy at detection limit levels.

Nanoslit design for ion conductivity gradient enhanced dielectrophoresis for ultrafast biomarker enrichment in physiological media

“…In fact, our prior work quantified this at 10 4 -10 5 -fold concentration enhancement in the preconcentration region 19 and $10 3 -fold enhancement in analyte binding at capture probe surface. 22,23 This is apparent from the sharp rise in fluorescence signal to saturation levels in Figure 4 . (iii)).…”

“…Our prior work has implemented this methodology within various sensing paradigms for improving biomarker sensitivity. 22,23 A cross-section view of the device geometry used within this work is schematically presented in Figure 1(a) (device images in S1, supplementary material), wherein glassy carbon modified Pt electrodes within the reservoirs leading to a microchannel on each side are used to initiate the discussed electrokinetic effects under a DC-offset (2 V/cm) AC field (70 V rms /cm) that is set at the critical frequency required to cause optimal biomarker nDEP, which is 1 MHz for streptavidin, 3 MHz for Neuropeptide Y (NPY), 22 and 4-6 MHz for Prostate Specific Antigen (PSA). 23 The microchannels (5 lm depth) are connected by slit-shaped channels of 200 nm depth (henceforth called nanoslit).…”

“…22,23 A cross-section view of the device geometry used within this work is schematically presented in Figure 1(a) (device images in S1, supplementary material), wherein glassy carbon modified Pt electrodes within the reservoirs leading to a microchannel on each side are used to initiate the discussed electrokinetic effects under a DC-offset (2 V/cm) AC field (70 V rms /cm) that is set at the critical frequency required to cause optimal biomarker nDEP, which is 1 MHz for streptavidin, 3 MHz for Neuropeptide Y (NPY), 22 and 4-6 MHz for Prostate Specific Antigen (PSA). 23 The microchannels (5 lm depth) are connected by slit-shaped channels of 200 nm depth (henceforth called nanoslit). Within each nanoslit (300 lm length/200 nm depth), a lateral constriction (optimized at 300 nm length and 40 nm gap) with a sharp surface charge non-uniformity is used to initiate ICP (see magnified top-view in Figure 1(b)), whereas no significant ICP occurs at the micro/ nanochannel interface due to the wide nanoslit entry points (30 lm width).…”

“…[20][21][22][23] Furthermore, the highly localized nature of DEP behavior, due to its dependence on rE 2 , limits its spatial extent. 13,24 Herein, by initiating ICP at lateral perm-selective constrictions of large surface charge non-uniformity, we create conductivity differences across the nanoslit length to enhance the spatial extent for biomarker depletion.…”

Quantifying Particle Motion under Force Fields in Microfluidic Systems by Image Tracking Methods

“…These techniques have been widely applied in trapping, characterizing and separating particles [1].Using electrical fields is very suitable for the precise control of bioparticles like bacteria [2], yeast, apoptosis, viruses, proteins [3] and DNA due to some advantages of good controllability, easy operation, high efficiency and small damage to the particles [1].…”