Immunoarray Measurements of Parathyroid Hormone-Related Peptides Combined with Other Biomarkers to Diagnose Aggressive Prostate Cancer.

Parathyroid hormone-related peptide (PTHrP) is related to bone metastasis and hypercalcemia in prostate and breast cancers and should be an excellent biomarker for aggressive forms of these cancers. Current clinical detection protocols for PTHrP are immunoradiometric assay and radioimmunoassay but are not sensitive enough to detect PTHrPs at early stages. We recently evaluated a prostate cancer biomarker panel, including serum monocyte differentiation antigen (CD-14), ETS-related gene protein, pigment epithelial-derived factor, and insulin-like growth factor-1, with promise for identifying aggressive prostate cancers. This panel predicted the need for patient biopsy better than PSA alone. In the present paper, we report an ultrasensitive microfluidic assay for PTHrPs and evaluate their diagnostic value and the value of including them with our prior biomarker panel to diagnose aggressive forms of prostate cancer. The immunoarray features screen-printed carbon sensor electrodes coated with 5 nm glutathione gold nanoparticles with capture antibodies attached. PTHrPs are bound to a secondary antibody attached to a polyhorseradish peroxidase label and delivered to the sensors to provide high sensitivity when activated by H2O2 and a mediator. We obtained an unprecedented 0.3 fg mL-1 limit of detection for PTHrP bioactive moieties PTHrP 1-173 and PTHrP 1-86. We also report the first study of PTHrPs in a large serum pool to identify aggressive malignancies. In assays of 130 human patient serum samples, PTHrP levels distinguished between aggressive and indolent prostate cancers with 83-91% clinical sensitivity and 78-96% specificity. Logistic regression identified the best predictive model as a combination of PTHrP 1-86 and vascular endothelial growth factor-D. PTHrP 1-173 alone also showed a high ability to differentiate aggressive and indolent cancers.

Prostate Cancer Diagnosis in the Clinic Using an 8-Protein Biomarker Panel.

The inability to distinguish aggressive from indolent prostate cancer is a longstanding clinical problem. Prostate specific antigen (PSA) tests and digital rectal exams cannot differentiate these forms. Because only ∼10% of diagnosed prostate cancer cases are aggressive, existing practice often results in overtreatment including unnecessary surgeries that degrade patients' quality of life. Here, we describe a fast microfluidic immunoarray optimized to determine 8-proteins simultaneously in 5 µL of blood serum for prostate cancer diagnostics. Using polymeric horseradish peroxidase (poly-HRP, 400 HRPs) labels to provide large signal amplification and limits of detection in the sub-fg mL-1 range, a protocol was devised for the optimization of the fast, accurate assays of 100-fold diluted serum samples. Analysis of 130 prostate cancer patient serum samples revealed that some members of the protein panel can distinguish aggressive from indolent cancers. Logistic regression was used to identify a subset of the panel, combining biomarker proteins ETS-related gene protein (ERG), insulin-like growth factor-1 (IGF-1), pigment epithelial-derived factor (PEDF), and serum monocyte differentiation antigen (CD-14) to predict whether a given patient should be referred for biopsy, which gave a much better predictive accuracy than PSA alone. This represents the first prostate cancer blood test that can predict which patients will have a high biopsy Gleason score, a standard pathology score used to grade tumors.

Printed Electrodes in Microfluidic Arrays for Cancer Biomarker Protein Detection.

Medical diagnostics is trending towards a more personalized future approach in which multiple tests can be digitized into patient records. In cancer diagnostics, patients can be tested for individual protein and genomic biomarkers that detect cancers at very early stages and also be used to monitor cancer progression or remission during therapy. These data can then be incorporated into patient records that could be easily accessed on a cell phone by a health care professional or the patients themselves on demand. Data on protein biomarkers have a large potential to be measured in point-of-care devices, particularly diagnostic panels that could provide a continually updated, personalized record of a disease like cancer. Electrochemical immunoassays have been popular among protein detection methods due to their inherent high sensitivity and ease of coupling with screen-printed and inkjet-printed electrodes. Integrated chips featuring these kinds of electrodes can be built at low cost and designed for ease of automation. Enzyme-linked immunosorbent assay (ELISA) features are adopted in most of these ultrasensitive detection systems, with microfluidics allowing easy manipulation and good fluid dynamics to deliver reagents and detect the desired proteins. Several of these ultrasensitive systems have detected biomarker panels ranging from four to eight proteins, which in many cases when a specific cancer is suspected may be sufficient. However, a grand challenge lies in engineering microfluidic-printed electrode devices for the simultaneous detection of larger protein panels (e.g., 50-100) that could be used to test for many types of cancers, as well as other diseases for truly personalized care.

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

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.

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 (< $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.

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.