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.
In addition to disease diagnostics, there is a need for biomarkers to predict severity of cancer therapy side effects such as oral mucositis. Oral mucositis is an inflammatory lesion of oral mucosa caused by high dose chemotherapy and/or radiation that is especially prevalent during oral cancer treatment. We describe here a semiautomated, modular microfluidic immunoarray optimized for ultrasensitive detection of pro-inflammatory cytokines involved in pathobiology of oral mucositis. Our goal is methodology to identify risk of mucositis early in oral cancer treatment, before the onset of lesions. Biomarkers include tumor necrosis factor (TNF-α), interleukin-6 (IL-6), interleukin-1β (IL-1β), and C-reactive protein (CRP). Protein analytes were captured from serum in a capture chamber by 1 μm magnetic beads coated with antibodies and enzyme labels. Beads are then transported downstream to a detection chamber containing an 8-sensor array coated with glutathione-gold nanoparticles (GSH-AuNP) and a second set of antibodies to capture the beads with analyte proteins. In this first application of the immunoarray to 4-protein multiplexed measurements, ultralow detection limits of 10–40 fg mL−1 in 5 μL serum were achieved for simultaneous detection in 30 min. Mass detection limits were 2.5–10 zeptomol, as few as 1500 molecules. Accuracy and diagnostic utility of the arrays was demonstrated by correlation of levels of the 4 biomarker proteins in serum from head and neck cancer patients with results from standard ELISA. This approach may lead to rapid, low-cost estimates of projected risk for severity of oral mucositis in cancer patients to enable improved therapeutic management.
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.
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