Nanomaterials and biomaterials in electrochemical arrays for protein detection

Abstract

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.

Figure 1

General features of ELISA-like immunoarray strategies to detect proteins demonstrating some uses of nanoparticles. Nanoparticles on the spot areas are linked to primary antibodies that capture the protein analytes (target antigens). After washing, a labeled detection antibody, or as illustrated here a multi-labeled (nano- or micro-) particle with attached detection antibodies, is added. This detection particle binds selectively to the captured analyte molecules. After additional washing with blocking agents to remove non-specific binding of the labeled species, electrical or optical detection is used to “count” the number of bound labels that is proportional to protein analyte concentration.

Figure 2

Detection antibody (Ab₂)-label conjugates used with electrochemical immunosensors for signal amplification.

Figure 3

Atomic force microscope images of immunosensor platforms: (a) High surface area SWCNT forest on silicon; (b) SWCNT forest with antibodies chemically attached to their ends; (c) a PDDA/gold nanoparticle (AuNP, 5 nm) bilayer on smooth mica; (d) phase contrast image of the same PDDA/AuNP bilayer; (e) anti-PSA antibodies attached onto carboxylate groups of the AuNP/PDDA bilayer. Reproduced with permission from ref. copyright Royal Society of Chemistry, 2005, and ref. copyright American Chemical Society 2009.

Figure 4

Amperometry in stirred solutions at −0.3 V and 2500 rpm after placing arrays in buffer containing 1 mM hydroquinone and then injecting H₂O₂ to 0.4 mM for 4-electrode SWNT immunoarrays incubated with antigen standards in 10 μL undiluted calf serum for 1.25 h followed by Ab₂-HRP (A & B) or Ab₂-streptavidin-HRP (C & D) in 10 μL 0.4% w/v casein and 0.05% Tween 20 PBS buffer: (A) PSA, (B) PSMA, (C) PF-4, and (D) IL-6. Reproduced with permission from ref. copyright American Chemical Society 2009.

Figure 5

Modular microfluidic device shown with three types of insertable 8-sensor arrays: (A) Microfluidic assay system featuring syringe pump, fixed-loop injector valve, and 70 μL PDMS channel that houses 8-sensor arrays and Ag/AgCl reference and Pt counter electrode wires; (B) two views of gold array featuring nL-scale wells (to enable trapping 1 μL droplets) fabricated from a gold CD by computer template printing and wet etching; right view is the full chip, left view depicts sensor elements in the wells; (C) screen-printed carbon array (Kanichi Ltd., UK) that has been coated with 5 nm gold nanoparticles (AuNP); (D) ink-jet printed gold array using an ink made from toluene and 4 nm alkyl-thiol coated AuNPs, followed by annealing and treating with a carboxyl-terminated alkyl thiol.

Figure 6

Conceptual illustration of off-line capture of proteins on magnetic HRP-MB-Ab₂ beads

Figure 7

Calibration of 8-sensor microfluidic immunoarray using off-line protein capture by multilabel HRP-MP-Ab₂ particles with 200,000 HRP labels/particle at −0.3 V vs. Ag/AgCl: (A) Amperometric peaks for PSA and IL-6 mixtures in serum generated by injecting 1 mM hydroquinone + 100 μM H₂O₂; (B) Calibration curves (C) Simultaneous determinations by the array and individual ELISAs for PSA and IL-6 in patient serum: 1–4 are from prostate cancer patients; 5 is cancer-free control. Reproduced with permission from ref. copyright Elsevier, 2011.

Figure 8

Oral cancer biomarker proteins detected in serum by an amperometric microfluidic array after incubating of Ab₂-MB-HRP-analytes in measurement chamber, then injecting mixture of H₂O₂ and hydroquinone: (A) duplicate responses in simultaneous measurements of standard mixture 10 fg mL⁻¹ IL-6, 15 fg mL⁻¹ IL-8, 25 fg mL⁻¹ VEGF, and 60 fg mL⁻¹ VEGF-C illustrating reproducibility, (B) responses to VEGF in mixtures of biomarker proteins (peaks for VEGF extracted from four-protein determinations and presented together), (C–F) Immunoarray calibrations of standard mixtures in calf serum for IL-6 (C), IL-8 (D), VEGF (E), VEGF-C (F), using background corrected peak currents. Standard deviations correspond to 2 sensors each on three separate arrays (n=6). Reproduced with permission from ref. copyright American Chemical Society 2012.

Figure 9

Sensor surfaces in arrays made from gold CDs: (A) Tapping mode AFM images of exposed bare gold CD-R surface, which retain original ridges of the CD; (B) AFM of anti-IL-6 capture antibody covalently linked to the gold CD-R surface after treatment with mercaptopropionic acid; (C) Detection of proteins bound on the array using streptavidin poly-HRP, which attaches to biotinylated anti-human IL-6 detection antibody bound to IL-6 on the sensor, then hydrogen peroxide and hydroquinone are injected to produce an amperometric peaks. Reproduced with permission from ref. copyright Royal Society of Chemistry, 2011.

Figure 10

SEM images of electrochemically rebuilt Au nanostructures before (a) and after (b) decorating them with Pt nanoparticles. (c) H₂O₂ sensitivity (at +0.5 V vs. Ag/AgCl reference) for various working electrodes (see text for details). (d) Cyclic voltammograms of freshly deposited and CV-activated Pt-black electrodes in 0.5 M H₂SO₄ at 50 mV/s. (e) H₂O₂ sensitivity vs. potential of activated nanostructured-Au/Pt electrodes along with background (in the absence of H₂O₂) and signal to noise (S/N) ratio. (f) Linear dynamic range (y = 46.7 + 23.1x, R² = 0.995) for H₂O₂ detection at 0.3 V vs. Ag/AgCl reference in 50 μL/min PBS flow. Inset shows staircase amperometric curve for four additions of 10 nM H₂O₂ at the limit of detection (LOD). Reproduced with permission from refs. and , copyright Elsevier, 2010 and 2011.

Figure 11

Schematic layout of (a) patterned electrodes together with PDMS microfluidicchannel. (b) Microlithographically defined Cr/Au electrode without the side-edge protection and thick gold before and (c) after application of positive bias on electrodes that dissolves the underlying Cr adhesion layer causing the formation of pinholes and edge delamination. (d) Cross-section design of Pt black electrodes, that are resistant to delamination as a result of side-edge protection via a photoresist insulator, and the removal of pinholes by the growth of a thick (5 μm) electroplated gold layer. Optical images of electrodes described in (b) (viewed from the back side) showing the gray layer of Cr before (e) and after (f) 21 cycles of CV from −0.5 to 0.8 V vs. Ag/AgCl, which causes partial Cr dissolution and exposes the gold over layer at the edges and pinholes. (g) SEM image of the top surface of CV-activated Pt-black electrode. Reproduced with permission from ref. copyright Elsevier, 2010.

Figure 12

Features of ECL microwell immunoarray: (A) 10 μL wells containing SWCNT forests in red on a 1 × 1 in. pyrolytic graphite chip (black). SWCNT forests with primary antibodies are attached to well bottoms. Wells are filled with sample solutions and incubated to capture analyte proteins. After washing, 100 nm RuBPY-silica-nanoparticles-Ab₂ are added and bind to the captured protein analytes. (B) The chip is placed in an open top electrochemical cell, 0.95 V vs. SCE is applied, and ECL is measured with a CCD camera. (C) AFM image showing the microwell polymer wall with adjacent SWCNT forest; (D) SWCNT forest in a microwell bottom after covalent attachment of anti-PSA antibody; (E) Optical micrograph of 4 spots on an array showing the light green hydrophobic polymer wall forming the microwells. Inset shows single well at higher magnification. Reproduced with permission from ref. , copyright American Chemical Society 2011.

Figure 13

ECL results for microwell immunoarray responses to PSA and IL-6 in serum mixtures at 0.95 V vs Ag/AgCl using 0.05% Tween 20 + 0.05% Triton-X 100 + 100 mM TprA at pH 7.5. RuBPY-silica nanoparticles (100 nm) used for detection had antibodies attached for both proteins: (A) (1) 5 ng mL⁻¹ PSA, (2) 1 ng mL⁻¹ IL-6:, (B) (1) 0.4 ng mL⁻¹ PSA, (2) 0.2 ng mL⁻¹ IL-6, (C) (1) 40 pg mL⁻¹ PSA, (2) 20 pg mL-1 IL-6, and (D) (1) 1 pg mL-1 PSA, (2) 0.25 pg mL-1 IL-6. In all images, controls are duplicate spots in the bottom row: (3) 0 pg mL-1 IL-6, and (4) 0 pg mL-1 PSA. Calibration curves using standards in calf serum are shown for (E) PSA, and (F) IL-6. Reproduced with permission from ref. , copyright American Chemical Society 2011.

Figure 14

Design of microfluidic ECL array for proteins: 1) syringe pump 2) injector valve, 3) switch valve to guide the sample to the desired channel, 4) tubing for inlet, 5) outlet, 6) poly(methylmethacrylate) (PMMA) plate, 7) Pt counter wire, 8) Ag/AgCl reference wire (wires are on the underside of PMMA plate), 9) polydimethylsiloxane (PDMS) channels, 10) pyrolytic graphite chip (PG) (2.5 cm × 2.5 cm) (black), surrounded by hydrophobic polymer (white) to make microwells. Bottoms of microwells (red rectangles) contain primary antibody-decorated SWCNT forests, 11) ECL label containing RuBPY-silica nanoparticles with cognate secondary antibodies are injected to the capture protein analytes previously bound to cognate primary antibodies. ECL is detected with a CCD camera. Reproduced with permission from ref. , copyright Springer-Verlag, 2013,

Figure 15

ECL results at 0.95 V vs. Ag/AgCl using 0.05% Tween 20 + 0.05% Triton-X 100 + 100 mM TPrA at pH 7.5 for microfluidic immunoarrays showing detection of PSA and IL-6 mixtures in serum; (a) Lanes: 1) 5 ng mL⁻¹ PSA and 2) 20 pg mL⁻¹ IL-6, (b) Lanes: 1) 100 pg mL⁻¹ PSA and 2) 200 pg mL⁻¹ IL-6, and (c) Lanes: 1) 100 fg mL⁻¹ PSA and 2) 10 fg mL⁻¹ IL-6. Bottom lanes in all images are controls 0 pg mL⁻¹ IL-6 and 0 pg mL⁻¹ PSA. Calibraton curves for (d) IL-6 and (e) PSA. ECL intensity plotted after subtracting relative ECL for protein-free controls. Error bars show standard deviations (n = 3). Reproduced with permission from ref. , copyright Springer-Verlag, 2013,

Scheme 1

Synthesis of 5 nm gold nanoparticles protected by glutathiones with outer carboxylate groups for eventual linkage of antibodies.