This paper describes fabrication of a novel electrochemiluminescent (ECL) immunosensor array featuring capture-antibody-decorated single-wall carbon nanotube forests (SWCNT) residing in the bottoms of 10 µL wells with hydrophobic polymer walls. Silica nanoparticles containing [Ru(bpy)3]2+ and secondary antibodies (RuBPY-silica-Ab2) are employed in this system for highly sensitive two-analyte detection. Antibodies to PSA and IL-6 were attached to the same RuBPY-silica-Ab2 particle. The array was fabricated by forming the wells on a conductive pyrolytic graphite chip (1 × 1 in.) with a single connection to a potentiostat to achieve ECL. The sandwich immunoassay protocol employs antibodies attached to SWCNTs in the wells to capture analyte proteins. Then RuBPY-silica-Ab2 is added to bind to the captured proteins. ECL is initiated in the microwells by electrochemical oxidation of tripropyl amine (TprA), which catalytically reduces [Ru(bpy)3]2+ in the 100 nm particles, and is measured with a coupled charged device (CCD) camera. Separation of the analytical spots by the hydrophobic wall barriers enabled simultaneous immunoassays for two proteins in a single sample without cross-contamination. Detection limit (DL) for prostate specific antigen (PSA) was 1 pg mL−1 and for interleukin-6 (IL-6) was 0.25 pg mL−1 (IL-6) in serum. Array determinations of PSA and IL-6 in patient serum were well-correlated with single-protein ELISAs. These microwell SWCNT immunoarrays provide a simple, sensitive approach to detection of two or more proteins.

Solid-state nanopores have been widely employed in sensing applications from Coulter counters to DNA sequencing devices. The analytical signal in such experiments is the change in ionic current flowing through the orifice caused by the large molecule or nanoparticle translocation through the pore. Conceptually similar nanopipette-based sensors can offer several advantages including the ease of fabrication and small physical size essential for local measurements and experiments in small spaces. This paper describes the first evaluation of nanopipettes with well characterized geometry for resistive-pulse sensing of Au nanoparticles (AuNP), nanoparticles coated with an allergen epitope peptide layer, and AuNP–peptide particles with bound antipeanut antibodies (IgY) on the peptide layer. The label-free signal produced by IgY-conjugated particles was strikingly different from those obtained with other analytes, thus suggesting the possibility of selective and sensitive resistive-pulse sensing of antibodies.

We report the first electrochemiluminescent immunosensor combining single-wall carbon nanotube forests with RuBPY-silica-secondary antibody nanoparticles for sensitive detection of cancer biomarker prostate specific antigen.The measurement of protein biomarkers holds significant promise for early cancer detection. 1 , 2 Detection of multiple protein biomarkers provides more reliable diagnostics than single biomarker measurements.1 -3 Proteins in serum can be measured with enzyme-linked immunosorbent assays (ELISA), 4 radioimmunoassay (RIA),5 electrophoretic immunoassay6 and mass spectrometry-based proteomics.7 However, there is a clear need for simple, rapid, sensitive, accurate and low cost protein detection suitable for point of care use.Electrochemiluminescence (ECL) is initiated when tris (2,2′-bipyridyl)ruthenium(II), [Ru-(bpy) 3 ] 2+ , or RuBPY, in its oxidized form reacts with a suitable sacrificial reductant, and has been used for detection of DNA and proteins. [8][9][10] Magnetic bead ECL methods have been commercialized. 11,12 Dye doped silica nanoparticles show promise as labels in ultrasensitive ECL bioassays. 13 RuBPY can be trapped inside mesoporous silica nanoparticles synthesized in water-in-oil (W/ O) microemulsions. 14 A single nanoparticle can encapsulate thousands of RuBPY to provide large signal amplification. Surfaces of the particles can be decorated with attached antibodies or proteins and arrays utilizing ECL can be fabricated using analytical spots on a simple conductive chip with a single connection to a power source and readout with a CCD camera, as we demonstrated with DNA-based toxicity screening arrays.9 This approach is simpler than the commercial bead-based ECL assays, which require sophisticated and expensive sample manipulation and measurement systems.15In this communication, we combine ECL nanoparticle labels with a single-wall carbon nanotube (SWCNT) forest platform in a sandwich immunoassay procedure for protein detection. The SWCNT forests feature self-assembled 20-30 nm long terminally carboxylated SWCNT standing in upright bundles on a thin Nafion-iron oxide layer on a pyrolytic graphite † Electronic supplementary information (ESI) available: Experimental details, Fig. S1 16 and provide a large conductive, functionalized surface area for attachment of capture antibodies in immunoassays.16b We have used SWCNT forests to build ultrasensitive amperometric immunosensors and detected PSA in serum using multiple enzyme labels.17 However, arrays using this strategy involve microfabricated multi-electrode chips as well as multi-electrode potentiostats, which are not necessary in ECL arrays.9In related work, [Ru-(bpy) 3 ] 2+ immobilized on composite films of disordered CNTs in combination with Nafion, 18a partially sulfonated polystyrene 18b and Eastman-AQ polymers 18c were used for ECL determination of tripropylamine 18 with a detection limit of 3 pM. 18c SWCNT forests with large carboxylated surface areas combined with immunoassays using RuBPY-silica-secondary ant...

We describe an electrochemiluminescence (ECL) immunoarray incorporated into a prototype microfluidic device for highly sensitive protein detection, and apply this system to accurate, sensitive measurements of prostate specific antigen (PSA) and interleukin-6 (IL-6) in serum. The microfluidic system employed three molded polydimethylsiloxane (PDMS) channels on a conductive pyrolytic graphite chip (PG) (2.5 × 2.5 cm) inserted into a machined chamber and interfaced with a pump, switching valve and sample injector. Each of the three PDMS channels encompasses three 3 μL analytical wells. Capture-antibody-decorated single-wall carbon nanotube (SWCNT) forests are fabricated in the bottom of the wells. The antigen is captured by these antibodies on the well bottoms. Then a RuBPY-silica-secondary antibody (Ab2) label is injected to bind to antigen on the array, followed by injection of sacrificial reductant tripropylamine (TPrA) to produce ECL. For detection, the chip is placed into an open-top ECL measuring cell, and the channels are in contact with electrolyte in the chamber. Potential applied at 0.95 V vs. SCE oxidizes TPrA to produce ECL by redox cycling the RuBPY species in the particles, and ECL light is measured by a CCD camera. This approach achieved ultralow detection limits (DL) of 100 fg mL-1 for PSA (9 zeptomol) and 10 fg mL-1 (1 zeptomol) for IL-6 in calf serum, a 10-25 fold improvement of a similar non-microfluidic array. PSA and IL-6 in synthetic cancer patient serum samples were detected in 1.1 h and results correlated well with single-protein ELISAs.

Point-of-care diagnostics based on multiplexed protein measurements face challenges of simple, automated, low-cost, and high-throughput operation with high sensitivity. Herein, we describe an automated, microprocessor-controlled microfluidic immunoarray for simultaneous multiplexed detection of small protein panels in complex samples. A microfluidic sample/reagent delivery cassette was coupled to a 30-microwell detection array to achieve sensitive detection of four prostate cancer biomarker proteins in serum. The proteins are prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), platelet factor-4 (PF-4), and interlukin-6 (IL-6). The six channel system is driven by integrated micropumps controlled by an inexpensive programmable microprocessor. The reagent delivery cassette and detection array feature channels made by precision-cut 0.8 mm silicone gaskets. Single-wall carbon nanotube forests were grown in printed microwells on a pyrolytic graphite detection chip and decorated with capture antibodies. The detection chip is housed in a machined microfluidic chamber with a steel metal shim counter electrode and Ag/AgCl reference electrode for electrochemiluminescent (ECL) measurements. The preloaded sample/reagent cassette automatically delivers antigen proteins, wash buffers, and ECL RuBPY-silica–antibody detection nanoparticles sequentially. An onboard microcontroller controls micropumps and reagent flow to the detection chamber according to a preset program. Detection employs tripropylamine, a sacrificial reductant, while applying 0.95 V vs Ag/AgCl. Resulting ECL light was measured by a CCD camera. Ultralow detection limits of 10–100 fg mL−1 were achieved in simultaneous detection of the four protein in 36 min assays. Results for the four proteins in prostate cancer patient serum gave excellent correlation with those from single-protein ELISA.