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

Abstract

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); r2 = 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.

Multiplexed Point-of-Care Testing – xPOCT

“…Array-based electrochemical systems depend on individually addressable microelectrode arrays 39 , 40 , 41 . Different biomolecules are directly immobilized on the electrodes and detected electrochemically via enzymatic reactions by potentiometry or amperometry.…”

“…Electrochemical approaches for microfluidic multiplexing enable the realization of compact analytical devices, which combine automated assay and measurement procedures along with rapid and sensitive detection. Herein, most of the systems employ separate electrochemical measurement cells within a channel network 40 , 71 , 72 . However, the application of a single electrochemical measurement cell with one or multiple working electrodes is highly beneficial for the system design of xPOCT devices.…”

Printed Electrodes in Microfluidic Arrays for Cancer Biomarker Protein Detection

“…Later, collaborations with Munge et al synthesized MBs with about 50,000 HRP enzymes, pushing LODs down to 1 fg mL −1 for interleukin 8 (IL-8) detection [ 94 ]. These studies were then extended for ultrasensitive single and multiplexed detection of cancer biomarker proteins on both SPEs and inkjet-printed electrodes coupled in microfluidic devices [ 31 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 ] ( Figure 6 ).…”

“…Otieno et al then modified this microfluidic device with an online protein capture chamber, where the analytes are separated from the serum in an isolated compartment with Mb-Ab 2 , before being introduced into the detection channel with the electrodes [ 99 ]. Otieno et al further detected parathyroid hormone-related peptide (PTHrP) fragments which act as biomarkers for bone metastasis of breast and prostate cancer obtaining LODs of 3 fg mL −1 ,1000-fold less than the traditionally used immunoradiometric assay (IRMA) [ 100 ].…”

“…Their findings indicate larger surface areas hinder reaching ultrasensitive measurements and suppression of the background signal can easily be achieved by minimal surface area [ 139 ]. We have already presented many examples of signal amplification by surface modification of SPE’s by nanoparticles [ 17 , 79 , 90 , 91 , 93 , 94 , 95 , 96 , 97 , 98 , 99 , 100 , 101 , 102 , 103 , 109 , 110 , 111 ]. A few more examples are included in this section.…”