P-Alkynyl functionalized benzazaphospholes as transmetalating agents.

Exposure of 10π-electron benzazaphosphole 1 to HCl, followed by nucleophilic substitution with the Grignard reagent BrMgCCPh afforded alkynyl functionalized 3 featuring an exocyclic -C[triple bond, length as m-dash]C-Ph group with an elongated P-C bond (1.7932(19) Å). Stoichiometric experiments revealed that treatment of trans-Pd(PEt3)2(Ar)(i) (Ar = p-Me (C) or p-F (D)) with 3 generated trans-Pd(PEt3)2(Ar)(CCPh) (Ar = p-Me (E) or p-F (F)), 5, which is the result of ligand exchange between P-I byproduct 4 and C/D, and the reductively eliminated product (Ar-C[triple bond, length as m-dash]C-Ph). Cyclic voltammetry studies showed and independent investigations confirmed 4 is also susceptible to redox processes including bimetallic oxidative addition to Pd(0) to give Pd(i) dimer 6-Pd2-(P(t-Bu)3)2 and reduction to diphosphine 7. During catalysis, we hypothesized that this unwanted reactivity could be circumvented by employing a source of fluoride as an additive. This was demonstrated by conducting a Sonogashira-type reaction between 1-iodotoluene and 3 in the presence of 10 mol% Na2PdCl4, 20 mol% P(t-Bu)Cy2, and 5 equiv. of tetramethylammonium fluoride (TMAF), resulting in turnover and the isolation of Ph-C[triple bond, length as m-dash]C-(o-Tol) as the major product.

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.

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.

Electrochemistry-based approaches to low cost, high sensitivity, automated, multiplexed protein immunoassays for cancer diagnostics.

Early detection and reliable diagnostics are keys to effectively design cancer therapies with better prognoses. The simultaneous detection of panels of biomarker proteins holds great promise as a general tool for reliable cancer diagnostics. A major challenge in designing such a panel is to decide upon a coherent group of biomarkers which have higher specificity for a given type of cancer. The second big challenge is to develop test devices to measure these biomarkers quantitatively with high sensitivity and specificity, such that there are no interferences from the complex serum or tissue matrices. Lastly, integrating all these tests into a technology that does not require exclusive training to operate, and can be used at point-of-care (POC) is another potential bottleneck in futuristic cancer diagnostics. In this article, we review electrochemistry-based tools and technologies developed and/or used in our laboratories to construct low-cost microfluidic protein arrays for the highly sensitive detection of a panel of cancer-specific biomarkers with high specificity which at the same time has the potential to be translated into POC applications.

Ultrasensitive microfluidic array for serum pro-inflammatory cytokines and C-reactive protein to assess oral mucositis risk in cancer patients.

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 semi-automated, modular microfluidic immunoarray optimized for ultrasensitive detection of pro-inflammatory cytokines involved in pathobiology of oral mucositis. Our goal is to methodologically identify risk of mucositis early in oral cancer treatment, before the onset of lesions. Biomarkers include tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6), interleukin-1ß (IL-1ß), 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 eight-sensor array coated with glutathione-coated 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 four-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 zmol, as few as 1500 molecules. Accuracy and diagnostic utility of the arrays were demonstrated by correlation of levels of the four 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.

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.

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).

Sensitive electrochemical immunosensor for matrix metalloproteinase-3 based on single-wall carbon nanotubes.

A novel electrochemical immunosensor for the detection of matrix metalloproteinase-3 (MMP-3), a cancer biomarker protein, based on vertically aligned single-wall carbon nanotube (SWCNT) arrays is presented. Detection was based on a sandwich immunoassay consisting of horseradish peroxidase (14-16 labels) conjugated to a secondary antibody and/or a polymer bead loaded with multi-enzyme labels. Performance was optimized by effective minimization of non-specific binding (NSB) events using Bovine serum albumin (BSA), Tween-20 and optimization of the primary antibody and secondary antibody concentrations. Results provided a detection limit of 0.4 ng mL(-1) (7.7 pM) for the 14-16 label sensor protocol and 4 pg mL(-1) (77 fM) using a multiply enzyme labeled polymeric bead amplification strategy in 10 microL of calf serum. This immunosensor based on SWCNT arrays offers great promise for a rapid, simple, cost-effective method for clinical screening of cancer biomarkers for point-of-care diagnosis.

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

Electrochemical immunosensors based on single wall nanotube (SWNT) forests and 5 nm glutathione-protected gold nanoparticles (GSH-AuNP) were developed and compared for the measurement of human cancer biomarker interleukin-6 (IL-6) in serum. Detection was based on sandwich immunoassays using multiple (14-16) horseradish peroxidase labels conjugated to a secondary antibody. Performance was optimized by effective blocking of non-specific binding (NSB) of the labels using bovine serum albumin. The GSH-AuNP immunosensor gave a detection limit (DL) of 10 pg mL(-1) IL-6 (500 amol mL(-1)) in 10 muL calf serum, which was 3-fold better than 30 pg mL(-1) found for the SWNT forest immunosensor for the same assay protocol. The GSH-AuNPs platform also gave a much larger linear dynamic range (20-4000 pg mL(-1)) than the SWNT system (40-150 pg mL(-1)), but the SWNTs had 2-fold better sensitivity in the low pg mL(-1) range.