Multiplexed electrochemical protein detection and translation to personalized cancer diagnostics

Abstract

Measuring diagnostic panels of multiple proteins promises a new, personalized approach to early detection and therapy of diseases like cancer. Levels of biomarker proteins in patient serum can provide a continually updated record of disease status. Research in electrochemical detection of proteins has produced exquisitely sensitive approaches. Most utilize ELISA-like sandwich immunoassays incorporating various aspects of nanotechnology. Several of these ultrasensitive methodologies have been extended to microfluidic multiplexed protein detection, but engineered solutions are needed to measure more proteins in a single device from a small patient sample such as a drop of blood or tissue lysate. To achieve clinical or point-of-care (POC) use, simplicity and low cost are essential. In multiplexed microfluidic immunoassays, required reagent additions and washing steps pose a significant problem calling for creative engineering. A grand challenge is to develop a general cancer screening device to accurately measure 50-100 proteins in a simple, cost-effective fashion. This will require creative solutions to simplified reagent addition and multiplexing.

Figure 1

Illustration of enzyme-linked electrochemical immunosensors using a sandwich assay format showing a nanostructured sensor element using a single enzyme label (path A) or a multi-labeled particle (path B). Primary antibodies (Ab1) on the sensor capture analyte protein from the sample. In the conventional approach (path A), an enzyme-labeled secondary antibody (Ab₂) is added to bind to the analyte protein on the sensor. In an ultrasensitive approach (path B), a magnetic bead with hundreds of thousands of antibodies and enzyme labels is added to bind to the analyte protein for signal amplification Addition of an enzyme substrate and electrochemical detection provides signals proportional to the amount of protein in the sample.

Figure 2

Receiver operating characteristic (ROC) curves for serum assays for (A) IL-6, AUC 0.86 (red line), IL-8 with AUC 0.83 (blue line), VEGF with AUC 0.95 (green line), VEGF-C with AUC 0.83 (brown line), and (B) mean normalized value for all 4 proteins, with AUC 0.96. Reprinted with permission from ref. 14. Copyright 2012 American Chemical Society.

Figure 3

Modular microfluidic immunoarray featuring on-line protein capture on magnetic beads. Upper left picture (B) shows stirred, magnetically controlled, reaction chamber where target proteins are captured on-line from samples by heavily-labeled HRP-antibody-magnetic beads to form antigen-bead bioconjugates, which are washed with magnet in place, then transferred by flow after magnet removal into detection chamber in assembled system at the bottom (A). Upper right (C) shows the microfluidic detection chamber featuring eight-sensor immunoarray in a 63 μL channel where bead flow can be controlled by a magnet (D).

Figure 4

Integrated mChip microfluidic device made by injection molding, including data on fluid handling of a POC ELISA-like assay for two antibodies: (a) Picture of microfluidic chip. Each chip can accommodate seven samples (one per channel), with molded holes for coupling of reagent-loaded tubes. (b) Scanning electron microscope image of a cross-section of microchannels, made of injection-molded plastic. Scale bar, 500 μm. (c) Transmitted light micrograph of channel meanders. Scale bar, 1 mm. (d) Schematic diagram of passive delivery of multiple reagents, which requires no moving parts on-chip. A preloaded sequence of reagents passes over a series of four detection zones, each characterized by dense meanders coated with capture proteins, before exiting the chip to a disposable syringe used to generate a vacuum for fluid actuation. (e) Illustration of biochemical reactions in detection zones at different immunoassay steps. The reduction of silver ions on gold nanoparticle–conjugated antibodies yields signals that can be read with low-cost optics (for quantification) or examined by eye. (f) Absorbance traces of a complete HIV-syphilis duplex test as reagent plugs pass through detection zones. High optical density (OD) is observed when air spacers pass through the detection zones, owing to increased refraction of light compared to in the liquid-filled channels. The train of reagents mimics the pipetting of reagents in and out of multiwell plates. This sample was evaluated (correctly against a reference standard) as HIV negative and syphilis positive. Ag = antigen. Reproduced with permission from ref. 19; Copyright © 2011, Nature Publishing Group.