Screen-Printed Soft-Nitrided Carbon Electrodes for Detection of Hydrogen Peroxide.

Nitrogen-doped carbon materials have garnered much interest due to their electrocatalytic activity towards important reactions such as the reduction of hydrogen peroxide. N-doped carbon materials are typically prepared and deposited on solid conductive supports, which can sometimes involve time-consuming, complex, and/or costly procedures. Here, nitrogen-doped screen-printed carbon electrodes (N-SPCEs) were fabricated directly from a lab-formulated ink composed of graphite that was modified with surface nitrogen groups by a simple soft nitriding technique. N-SPCEs prepared from inexpensive starting materials (graphite powder and urea) demonstrated good electrocatalytic activity towards hydrogen peroxide reduction. Amperometric detection of H2O2 using N-SPCEs with an applied potential of -0.4 V (vs. Ag/AgCl) exhibited good reproducibility and stability as well as a reasonable limit of detection (2.5 µM) and wide linear range (0.020 to 5.3 mM).

Ultrathin Graphene-Protein Supercapacitors for Miniaturized Bioelectronics.

Nearly all implantable bioelectronics are powered by bulky batteries which limit device miniaturization and lifespan. Moreover, batteries contain toxic materials and electrolytes that can be dangerous if leakage occurs. Herein, an approach to fabricate implantable protein-based bioelectrochemical capacitors (bECs) employing new nanocomposite heterostructures in which 2D reduced graphene oxide sheets are interlayered with chemically modified mammalian proteins, while utilizing biological fluids as electrolytes is described. This protein-modified reduced graphene oxide nanocomposite material shows no toxicity to mouse embryo fibroblasts and COS-7 cell cultures at a high concentration of 1600 µg mL-1 which is 160 times higher than those used in bECs, unlike the unmodified graphene oxide which caused toxic cell damage even at low doses of 10 µg mL-1. The bEC devices are 1 µm thick, fully flexible, and have high energy density comparable to that of lithium thin film batteries. COS-7 cell culture is not affected by long-term exposure to encapsulated bECs over 4 d of continuous charge/discharge cycles. These bECs are unique, protein-based devices, use serum as electrolyte, and have the potential to power a new generation of long-life, miniaturized implantable devices.

Fe3O4 nanoparticles on graphene oxide sheets for isolation and ultrasensitive amperometric detection of cancer biomarker proteins.

Ultrasensitive mediator-free electrochemical detection for biomarker proteins was achieved at low cost using a novel composite of Fe3O4 nanoparticles loaded onto graphene oxide (GO) nano-sheets (Fe3O4@GO). This paramagnetic Fe3O4@GO composite (1µm size range) was decorated with antibodies against prostate specific antigen (PSA) and prostate specific membrane antigen (PSMA), and then used to first capture these biomarkers and then deliver them to an 8-sensor detection chamber of a microfluidic immunoarray. Screen-printed carbon sensors coated with electrochemically reduced graphene oxide (ERGO) and a second set of antibodies selectively capture the biomarker-laden Fe3O4@GO particles, which subsequently catalyze hydrogen peroxide reduction to detect PSA and PSMA. Accuracy was confirmed by good correlation between patient serum assays and enzyme-linked immuno-sorbent assays (ELISA). Excellent detection limits (LOD) of 15 fg/mL for PSA and 4.8 fg/mL for PSMA were achieved in serum. The LOD for PSA was 1000-fold better than the only previous report of PSA detection using Fe3O4. Dynamic ranges were easily tunable for concentration ranges encountered in serum samples by adjusting the Fe3O4@GO Concentration. Reagent cost was only $0.85 for a single 2-protein assay.

3D-printed bioanalytical devices.

While 3D printing technologies first appeared in the 1980s, prohibitive costs, limited materials, and the relatively small number of commercially available printers confined applications mainly to prototyping for manufacturing purposes. As technologies, printer cost, materials, and accessibility continue to improve, 3D printing has found widespread implementation in research and development in many disciplines due to ease-of-use and relatively fast design-to-object workflow. Several 3D printing techniques have been used to prepare devices such as milli- and microfluidic flow cells for analyses of cells and biomolecules as well as interfaces that enable bioanalytical measurements using cellphones. This review focuses on preparation and applications of 3D-printed bioanalytical devices.

Electrochemiluminescence at Bare and DNA-Coated Graphite Electrodes in 3D-Printed Fluidic Devices.

Clear plastic fluidic devices with ports for incorporating electrodes to enable electrochemiluminescence (ECL) measurements were prepared using a low-cost, desktop three-dimensional (3D) printer based on stereolithography. Electrodes consisted of 0.5 mm pencil graphite rods and 0.5 mm silver wires inserted into commercially available 1/4 in.-28 threaded fittings. A bioimaging system equipped with a CCD camera was used to measure ECL generated at electrodes and small arrays using 0.2 M phosphate buffer solutions containing tris(2,2'-bipyridyl)dichlororuthenium(II) hexahydrate ([Ru(bpy)3]2+) with 100 mM tri-n-propylamine (TPA) as the coreactant. ECL signals produced at pencil graphite working electrodes were linear with respect to [Ru(bpy)3]2+ concentration for 9-900 µM [Ru(bpy)3]2+. The detection limit was found to be 7 µM using the CCD camera with exposure time set at 10 s. Electrode-to-electrode ECL signals varied by ±7.5%. Device performance was further evaluated using pencil graphite electrodes coated with multilayer poly(diallyldimethylammonium chloride) (PDDA)/DNA films. In these experiments, ECL resulted from the reaction of [Ru(bpy)3]3+ with guanines of DNA. ECL produced at these thin-film electrodes was linear with respect to [Ru(bpy)3]2+ concentration from 180 to 800 µM. These studies provide the first demonstration of ECL measurements obtained using a 3D-printed closed-channel fluidic device platform. The affordable, high-resolution 3D printer used in these studies enables easy, fast, and adaptable prototyping of fluidic devices capable of incorporating electrodes for measuring ECL.

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.

Resistive-Pulse Measurements with Nanopipettes: Detection of Vascular Endothelial Growth Factor C (VEGF-C) Using Antibody-Decorated Nanoparticles.

Quartz nanopipettes have recently been employed for resistive-pulse sensing of Au nanoparticles (AuNP) and nanoparticles with bound antibodies. The analytical signal in such experiments is the change in ionic current caused by the nanoparticle translocation through the pipette orifice. This paper describes resistive-pulse detection of cancer biomarker (Vascular Endothelial Growth Factor-C, VEGF-C) through the use of antibody-modified AuNPs and nanopipettes. The main challenge was to differentiate between AuNPs with attached antibodies for VEGF-C and antigen-conjugated particles. The zeta-potentials of these types of particles are not very different, and, therefore, carefully chosen pipettes with well-characterized geometry were necessary for selective detection of VEGF-C.

3D-Printed Fluidic Devices for Nanoparticle Preparation and Flow-Injection Amperometry Using Integrated Prussian Blue Nanoparticle-Modified Electrodes.

A consumer-grade fused filament fabrication (FFF) 3D printer was used to construct fluidic devices for nanoparticle preparation and electrochemical sensing. Devices were printed using poly(ethylene terephthalate) and featured threaded ports to connect polyetheretherketone (PEEK) tubing via printed fittings prepared from acrylonitrile butadiene styrene (ABS). These devices included channels designed to have 800 µm × 800 µm square cross sections and were semitransparent to allow visualization of the solution-filled channels. A 3D-printed device with a Y-shaped mixing channel was used to prepare Prussian blue nanoparticles (PBNPs) under flow rates of 100 to 2000 µL min(-1). PBNPs were then attached to gold electrodes for hydrogen peroxide sensing. 3D-printed devices used for electrochemical measurements featured threaded access ports into which a fitting equipped with reference, counter, and PBNP-modified working electrodes could be inserted. PBNP-modified electrodes enabled amperometric detection of H2O2 in the 3D-printed channel by flow-injection analysis, exhibiting a detection limit of 100 nM and linear response up to 20 µM. These experiments show that a consumer-grade FFF printer can be used to fabricate low-cost fluidic devices for applications similar to those that have been reported with more expensive 3D-printing methods.

Low-cost photolithographic fabrication of nanowires and microfilters for advanced bioassay devices.

Integrated microfluidic devices with nanosized array electrodes and microfiltration capabilities can greatly increase sensitivity and enhance automation in immunoassay devices. In this contribution, we utilize the edge-patterning method of thin aluminum (Al) films in order to form nano- to micron-sized gaps. Evaporation of high work-function metals (i.e., Au, Ag, etc.) on these gaps, followed by Al lift-off, enables the formation of electrical uniform nanowires from low-cost, plastic-based, photomasks. By replacing Al with chromium (Cr), the formation of high resolution, custom-made photomasks that are ideal for low-cost fabrication of a plurality of array devices were realized. To demonstrate the feasibility of such Cr photomasks, SU-8 micro-pillar masters were formed and replicated into PDMS to produce micron-sized filters with 3-4 µm gaps and an aspect ratio of 3. These microfilters were capable of retaining 6 µm beads within a localized site, while allowing solvent flow. The combination of nanowire arrays and micro-pillar filtration opens new perspectives for rapid R&D screening of various microfluidic-based immunoassay geometries, where analyte pre-concentration and highly sensitive, electrochemical detection can be readily co-localized.

Nanomaterials and biomaterials in electrochemical arrays for protein detection.

Nanomaterials and biomaterials are important components of new electrochemical arrays designed for sensitive detection of proteins in biological fluids. Such multiplexed protein arrays are predicted to have an important future in personalized medical diagnostics, especially for cancer and heart disease. Sandwich immunoassays for proteins benefit greatly in sensitivity from the use of nanostructured sensor surfaces and multilabeled detection strategies involving nano- or microparticles. In these assays, capture agents such as antibodies or aptamers are attached to sensor surfaces in the array. Target proteins with large binding constants for the affinity agents are captured from liquid samples with high efficiency, either on the sensors or on magnetic bioconjugate particles decorated with many copies of labels and antibodies. After target proteins are captured on the sensor surfaces, the labels are detected by electrochemical techniques. This feature article begins with an overview of the recent history of nanoparticles in electrochemical protein sensors, then moves on to specific examples from our own laboratories. We discuss fabrication of nanostructured sensors and arrays with the aim of multiplexed detection as well as reusability. Following this, we describe systems that integrate particle-based protein sensing with microfluidics for multiplexed protein detection. We end with predictions on the diagnostic future of protein detection.

Electroosmotic flow rectification in pyramidal-pore mica membranes.

We demonstrate here a new electrokinetic phenomenon, Electroosmotic flow (EOF) rectification, in synthetic membranes containing asymmetric pores. Mica membranes with pyramidally shaped pores prepared by the track-etch method were used. EOF was driven through these membranes by using an electrode in solutions on either side to pass a constant ionic current through the pores. The velocity of EOF depends on the polarity of the current. A high EOF velocity is obtained when the polarity is such that EOF is driven from the larger base opening to the smaller tip opening of the pore. A smaller EOF velocity is obtained when the polarity is reversed such that EOF goes from tip to base. We show that this rectified EOF phenomenon is the result of ion current-rectification observed in such asymmetric-pore membranes.

Resistive-pulse studies of proteins and protein/antibody complexes using a conical nanotube sensor.

There is increasing interest in using nanopores in synthetic membranes as resistive-pulse sensors for molecular and macromolecule analytes. In general, this method entails measuring current pulses associated with translocation of the analyte through the nanopore sensor element. A key challenge for this sensing paradigm is building selectivity into the protocol so that the current pulses for the target analyte can be distinguished from current pulses for other species that might be present in the sample. We show here that this can be accomplished with a protein analyte by adding to the solution an antibody that selectively binds the protein. We demonstrate this concept using bovine serum albumin (BSA) and a Fab fragment from a BSA-binding polyclonal antibody. Because the complex formed upon binding of the Fab to BSA is larger than the free BSA molecule, the current-pulse signature for the BSA/Fab complex can be easily distinguished from the free BSA. Furthermore, the BSA/Fab pulses can be easily distinguished from the pulses obtained for the free Fab and from pulses obtained for a control protein that does not bind to the Fab. Finally, we also show that the current-pulse signature for the BSA/Fab complex can provide information about the size and stoichiometry of the complex.