Colleen E. Krause

Inkjet-printed gold nanoparticle electrochemical arrays on plastic. Application to immunodetection of a cancer biomarker protein

Electrochemical detection combined with nanostructured sensor surfaces offers potentially low-cost, high-throughput solutions for detection of clinically significant proteins. Inkjet printing offers an inexpensive non-contact fabrication method for microelectronics that is easily adapted for incorporating into protein immunosensor devices. Herein we report the first direct fabrication of inkjet-printed gold nanoparticle arrays, and apply them to electrochemical detection of the cancer biomarker interleukin-6 (IL-6) in serum. The gold nanoparticle ink was printed on a flexible, heat resistant polyimide Kapton substrate and subsequently sintered to create eight-electrode arrays costing <0.2 euro per array. The inkjet-printed working electrodes had reproducible surface areas with RSD <3%. Capture antibodies for IL-6 were linked onto the eight-electrode array, and used in sandwich immunoassays. A biotinylated secondary antibody with 16-18 horseradish peroxidase labels was used, and detection was achieved by hydroquinone-mediated amperometry. The arrays provided a clinically relevant detection limit of 20 pg mL(-1) in calf serum, sensitivity of 11.4 nA pg(-1) cm(-2), and a linear dynamic range of 20-400 pg mL(-1).

On-line protein capture on magnetic beads for ultrasensitive microfluidic immunoassays of cancer biomarkers.

Accurate, sensitive, multiplexed detection of biomarker proteins holds significant promise for personalized cancer diagnostics. Here we describe the incorporation of a novel on-line chamber to capture cancer biomarker proteins on magnetic beads derivatized with 300,000 enzyme labels and 40,000 antibodies into a modular microfluidic immunoarray. Capture and detection chambers are produced from PDMS on machined molds and do not require lithography. Protein analytes are captured from serum or other biological samples in the stirred capture chamber on the beads held in place magnetically. The beads are subsequently washed free of sample components, and wash solutions sent to waste. Removal of the magnet and valve switching sends the magnetic bead-protein bioconjugates into a detection chamber where they are captured on 8 antibody-decorated gold nanoparticle-film sensors and detected amperometrically. Most steps in the immunoassay including protein capture, washing and measurement are incorporated into the device. In simultaneous assays, the microfluidic system gave ultralow detection limits of 5 fg mL(-1) for interleukin-6 (IL-6) and 7 fg mL(-1) for IL-8 in serum. Accuracy was demonstrated by measuring IL-6 and IL-8 in conditioned media from oral cancer cell lines and showing good correlations with standard ELISAs. The on-line capture chamber facilitates rapid, sensitive, repetitive protein separation and measurement in 30 min in a semi-automated system adaptable to multiplexed protein detection.

Rapid Microfluidic Immunoassays of Cancer Biomarker Proteins Using Disposable Inkjet-Printed Gold Nanoparticle Arrays

Conventional protein detection methods such as enzyme-linked immunosorbent assays (ELISA) often take many hours to complete and usually only apply to one protein at a time. More rapid, multiplexed methods are needed for point-of-care (POC) and surgical applications in future personalized cancer diagnostics and therapy. This paper describes a low-cost inkjet-printed gold nanoparticle (AuNP) sensor chip integrated into a simple microfluidic immunoarray to achieve detection of two cancer biomarker proteins in 5 μL samples in 8 min. Magnetic beads of 1 μm diameter derivatized with ∼300 000 enzyme labels and thousands of antibodies were used to capture the biomarker proteins from samples. The beads with captured proteins are then injected into the microfluidic system and captured by antibodies on nanostructured sensor elements to provide high sensitivity and ultralow detection limits (DL). For assay times of 45 mins, DLs were 78 fg mL−1 for interleukin-6 (IL-6) and 19 fg mL−1 for interleukin-8 (IL-8). Decreasing assay time to 8 min provided clinically relevant DLs of 5 pg mL−1. Accuracy was demonstrated by determining IL-6 and IL-8 in conditioned media from head and neck squamous cell carcinoma (HNSCC) cells and comparing results to those from standard single-protein ELISAs. Results suggest that this device can be employed for rapid detection of a wide range of disease-related proteins in clinical applications.

Cancer Diagnostics via Ultrasensitive Multiplexed Detection of Parathyroid Hormone-Related Peptides with a Microfluidic Immunoarray

Parathyroid hormone-related peptide (PTHrP) is recognized as the major causative agent of humoral hypercalcemia of malignancy (HHM). The paraneoplastic PTHrP has also been implicated in tumor progression and metastasis of many human cancers. Conventional PTHrP detection methods like immunoradiometric assay (IRMA) lack the sensitivity required to measure target peptide levels prior to the development of hypercalcemia. In general, sensitive, multiplexed peptide measurement by immunoassay represents challenges that we address in this paper. We describe here the first ultrasensitive multiplexed peptide assay to measure intact PTHrP 1-173 as well as circulating N-terminal and C-terminal peptide fragments. This versatile approach should apply to almost any collection of peptides that are long enough to present binding sites for two antibodies. To target PTHrP, we employed a microfluidic immunoarray featuring a chamber for online capture of the peptides from serum onto magnetic beads decorated with massive numbers of peptide-specific antibodies and enzyme labels. Magnetic bead-peptide conjugates were then washed and sent to a detection chamber housing an antibody-modified 8-electrode array fabricated by inkjet printing of gold nanoparticles. Limits of detection (LODs) of 150 aM (∼1000-fold lower than IRMA) in 5 μL of serum were achieved for simultaneous detection of PTHrP isoforms and peptide fragments in 30 min. Good correlation for patient samples was found with IRMA (n = 57); r(2) = 0.99 assaying PTHrP 1-86 equiv fragments. Analysis by a receiver operating characteristic (ROC) plot gave an area under the curve of 0.96, 80-83% clinical sensitivity, and 96-100% clinical specificity. Results suggest that PTHrP1-173 isoform and its short C-terminal fragments are the predominant circulating forms of PTHrP. This new ultrasensitive, multiplexed assay for PTHrP and fragments is promising for clinical diagnosis, prognosis, and therapeutic monitoring from early to advanced stage cancer patients and to examine underlying mechanisms of PTHrP overproduction.

Disposable inkjet-printed electrochemical platform for detection of clinically relevant HER-2 breast cancer biomarker

Rapidly fabricated, disposable sensor platforms hold tremendous promise for point-of-care detection. Here, we present an inexpensive (<

Bioconjugation of Antibodies and Enzyme Labels onto Magnetic Beads.

0.25) fully inkjet printed electrochemical sensor with integrated counter, reference, and working electrodes that is easily scalable for commercial fabrication. The electrochemical sensor platform featured an inkjet printed gold working 8-electrode array (WEA) and counter electrode (CE), along with an inkjet -printed silver electrode that was chlorinated with bleach to produce a Ag/AgCl quasi-reference electrode (RE). As proof of concept, the electrochemical sensor was successfully applied for detection of clinically relevant breast cancer biomarker Human Epidermal Growth Factor Receptor 2 (HER-2). Capture antibodies were bound to a chemically modified surface on the WEA and placed into a microfluidic device. A full sandwich immunoassay was constructed following a simultaneous injection of target protein, biotinylated antibody, and polymerized horseradish peroxide labels into the microfluidic device housing the WEA. With an ultra fast assay time, of only 15mins a clinically relevant limit of detection of 12pgmL-1 was achieved. Excellent reproducibility and sensitivity were observed through recovery assays preformed in human serum with recoveries ranging from 76% to 103%. These easily fabricated and scalable electrochemical sensor platforms can be readily adapted for multiplex detection following this rapid assay protocol for cancer diagnostics.

Electrochemical Immunosensors for Interleukin-6. Comparison of Carbon Nanotube Forest and Gold Nanoparticle platforms.

Immunoassays employ antibodies and labels to capture and detect target macromolecular analytes, often from complex sample matrices such as serum, plasma, or saliva. The high affinity and specificity of antibody-antigen interactions makes immunoassays critically important analytical techniques for clinical diagnostics as well as other research applications in the areas of pharmaceutical and environmental analysis. Integration of magnetic beads (MBs) into immunoassays and other bioanalytical methodologies is a valuable approach to allow efficient target capture, enrichment, and convenient separation. In addition, large signal amplification can be achieved by preconcentration of the target and by attaching many thousands of enzyme labels to the MBs. These features have enabled MB-based biosensors to achieve ultra-low detection limits needed for advanced clinical diagnostics that are challenging or impossible using traditional immunoassays. MBs are employed either as mobile substrates for target analyte capture, as detection labels (or label carriers), or simultaneously as substrates and labels. For optimal assay performance, it is crucial to apply an easy, efficient, and robust bead-probe conjugation protocol, and to thoroughly characterize the bioconjugated products. Herein, we describe methods used in our laboratory to functionalize MBs with antibodies and enzyme labels for ultrasensitive detection of protein analytes. We also present detailed strategies for characterizing the MB bioconjugates.