Sub-zeptomole Detection of Biomarker Proteins Using a Microfluidic Immunoarray with Nanostructured Sensors

Abstract

We report here a low-cost electrochemical immunoarray with unprecedented sensitivity in the sub-zeptomole range with up to 5 log-decades dynamic range for accurate, multiplexed protein determinations. The microfluidic array features eight carbon sensors coated with a dense layer of 5 nm gold-nanoparticles derivatized with primary antibodies. Analyte proteins are captured by secondary antibody-poly-HPR (horseradish peroxidase) bioconjugates containing 400 HRP enzyme labels, with amplified amperometric peaks developed using H2O2 activator and hydroquinone mediator. Prostate cancer biomarkers prostate specific antigen (PSA), vascular endothelial growth factor-D (VEGF-D), ETS-related gene protein (ERG), and insulin-like growth factor-1 (IGF-1) were measured simultaneously with sub-fg/mL LODs (0.08–0.22 zmol). These proteins were determined in serum of postprostatectomy cancer patients which had much lower levels than prostate cancer patients without surgery. This immunoassay protocol makes thousands of low-abundance proteins accessible to quantitative measurements down to zeptomole levels.

Printed Electrodes in Microfluidic Arrays for Cancer Biomarker Protein Detection

“…Typically, after the immobilization of Ab 1 and the analytes captured, the electrodes are washed by detergent and/or an NSB blocking protein, such as bovine serum albumin (BSA) or casein, to reduce the NSB that greatly decreases the signal/noise (S/N) ratio [ 9 , 10 , 11 , 17 , 24 ]. ( Scheme 1 F) NSB arises when the Ab 2 molecules bind to non-antigen sites of the electrodes, providing a signal which is not proportionate to the analyte concentration, increasing the LOD and damaging the sensitivity.…”

“…( Scheme 1 F) NSB arises when the Ab 2 molecules bind to non-antigen sites of the electrodes, providing a signal which is not proportionate to the analyte concentration, increasing the LOD and damaging the sensitivity. Casein and BSA are used to prevent NSB by sterically blocking the non-analyte sites, whereas detergent such as phosphate-buffered saline-Tween 20 (PBS-T20) or Tris buffer solution (TBS), with sodium dodecyl sulfate (SDS) or Triton 100-X, help to wash away weakly-bound Ab 2 on non-analyte sites [ 17 , 56 ]. Another approach on reduction of NSB is through chemical attachments of the electrode surface to reduce protein adsorption through (a) polymerization strategies (polyethylene glycol (PEG), conducting polymers), (b) modification of allotropic carbons (carbon nanotubes), (c) sol-gel modification, (d) surface modification by diazonium salts, (e) metal nanoparticles (magnetic beads, gold and silver nanoparticles), (f) self-assembled monolayers (SAMs) [ 52 ].…”

“…Approaches include (a) redox cycling by using reversible redox couples regenerated electrochemically, chemically, or enzymatically (discussed in detail by Yang et al) [ 48 ]; (b) multiple labeling such as multi-enzyme nanoparticles or polymers that provide a manifold of electrochemical events per bound analyte [ 48 ]. Other approaches include secondary antibody tags with dissolvable nanoparticles and multi-enzyme Ab 2 conjugates ( Figure 3 ) [ 9 , 10 , 11 , 17 , 48 ].…”

“…Generally, these multi-enzyme tags require a mediator for their effective functionality as the bulky particles provide a steric and distance barrier for direct electron transfer between the enzyme and the sensor electrode surface. Hydroquinone is a frequently used mediator coupled with H 2 O 2 that is being used on SPE systems [ 17 , 48 , 82 , 84 ].…”

“…Despite significant research gains in this area, only a handful of protein cancer biomarkers and no protein panels are approved by the US Food and Drug Administration (FDA) [ 3 , 5 , 14 , 15 ] ( Table 1 ) for clinical practice. This discrepancy between research and clinical practice is due to three main factors: (a) Lag in development of point-of-care (POC) devices for detection of panels of biomarkers [ 5 , 9 , 13 ] (b) lack of sufficient National Institutes of Health (NIH) funding for translational research in development and validation of effective biomarker panels, and (c) inability to detect rare biomarkers due to poor sensitivity for low concentration biomarkers coupled in a panel with more abundant proteins [ 16 , 17 , 18 , 19 ]. Detection of a panel of biomarkers enables better diagnostic accuracy than single proteins.…”