Immunoassays make use of highly specific antigen-antibody binding and provide sensitive ways to detect a wide range of biomacromolecules, bacteria, viruses, and small molecules. There are a range of types of immunoassay systems including single analyte sensors, 96well plate formats, arrays, microfluidic sensors, microfluidic arrays, etc. A big target is encompassed by medical diagnostic biomarkers, which are "molecules that can be measured objectively as indicators of normal or disease processes and responses to therapeutic intervention". 1 Accurate, low-cost measurements of multiple proteins are major applications of immunoarrays that are critical for future early detection and monitoring of cancer and other diseases. Multiplexing is very important, since panels of biomarker proteins, as opposed to single biomarkers, are required to provide sufficient information content for reliable disease diagnostics.

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.

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.

While the technology is relatively new, low-cost 3D printing has impacted many aspects of human life. 3D printers are being used as manufacturing tools for a wide variety of devices in a spectrum of applications ranging from diagnosis to implants to external prostheses. The ease of use, availability of 3D-design software and low cost has made 3D printing an accessible manufacturing and fabrication tool in many bioanalytical research laboratories. 3D printers can print materials with varying density, optical character, strength and chemical properties that provide the user with a vast array of strategic options. In this review, we focus on applications in biomedical diagnostics and how this revolutionary technique is facilitating the development of low-cost, sensitive, and often geometrically complex tools. 3D printing in the fabrication of microfluidics, supporting equipment, and optical and electronic components of diagnostic devices is presented. Emerging diagnostics systems using 3D bioprinting as a tool to incorporate living cells or biomaterials into 3D printing is also reviewed.

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.