A high throughput electrochemiluminescent (ECL) chip was fabricated and integrated into a fluidic system for screening toxicity-related chemistry of drug and pollutant metabolites. The chip base is conductive pyrolytic graphite onto which are printed 64 microwells capable of holding one-µL droplets. Films combining DNA, metabolic enzymes and an ECL-generating ruthenium metallopolymer (RuIIPVP) are fabricated in these microwells. The system runs metabolic enzyme reactions, and subsequently detects DNA damage caused by reactive metabolites. The performance of the chip was tested by measuring DNA damage caused by metabolites of the well-known procarcinogen benzo[a]pyrene (B[a]P). Liver microsomes and cytochrome P450 (cyt P450) enzymes were used with and without epoxide hydrolase (EH), a conjugative enzyme required for multi-enzyme bioactivation of B[a]P. DNA adduct formation was confirmed by determining specific DNA-metabolite adducts using similar films of DNA/enzyme on magnetic bead biocolloid reactors, hydrolyzing the DNA, and analyzing by capillary liquid chromatography-mass spectrometry (CapLC-MS/MS). The fluidic chip was also used to measure IC50-values of inhibitors of cyt P450s. All results show good correlation with reported enzyme activity and inhibition assays.

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.

Reactive oxygen species (ROS) oxidize guanosines in DNA to form 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG), a biomarker for oxidative stress. Herein we describe a novel 64-microwell electrochemiluminescent (ECL) array enabling sensitive multiplexed detection of 8-oxodG in ds-DNA without hydrolysis. Films of Nafion and reduced graphene oxide containing ECL dye [Os(bpy)2(phen-benz-COOH)]2+ (OsNG, {bpy= 2,2′-bipyridine and phen-benz-COOH = (4-(1,10-phenanthrolin-6-yl) benzoic acid)}) were assembled into microwells on a pyrolytic graphite wafer to detect 8-oxodG in oligonucleotides by electrochemiluminescence (ECL). DNA oxidation by Fenton’s reagent or by ROS formation during redox cycles involving NADPH, CuII, and model metabolites was monitored. UPLC-MS/MS of oxidized DNA samples were used for calibration. Detection limit for the fluidic arrays was one 8-oxodG per 670 intact nucleobases, or 0.15%. The method is sensitive enough to evaluate DNA oxidation from biologically relevant ROS-generating reactions of CuII, NADPH, and model metabolites.

We report herein a novel pipet-based "ELISA in a tip" as a new versatile diagnostic tool featuring better sensitivity, shorter incubation time, accessibility, and low sample and reagent volumes compared to traditional ELISA. Capture and analysis of data by a cell phone facilitates electronic delivery of results to health care providers. Pipette tips were designed and 3D printed as adapters to fit most commercial 50−200 μL pipettes. Capture antibodies (Ab 1 ) are immobilized on the inner walls of the pipet tip, which serves as the assay compartment where samples and reagents are moved in and out by pipetting. Signals are generated using colorimetric or chemiluminescent (CL) reagents and can be quantified using a cell phone, CCD camera, or plate reader. We utilized pipet-tip ELISA to detect four cancer biomarker proteins with detection limits similar to or lower than microplate ELISAs at 25% assay cost and time. Recoveries of these proteins from spiked human serum were 85−115% or better, depending slightly on detection mode. Using CCD camera quantification of CL with femto-luminol reagent gave limits of detection (LOD) as low as 0.5 pg/mL. Patient samples (13) were assayed for 3 biomarker proteins with results well correlated to conventional ELISA and an established microfluidic electrochemical immunoassay.

Understanding of the synthesis kinetics and our ability to modulate medium conditions allowed us to generate nanoparticles via an ultra-fast process. The synthesis medium is kept quite simple with tetraethyl orthosilicate (TEOS) as precursor and 50% ethanol and sodium hydroxide catalyst. Synthesis is performed under gentle conditions at 20 °C for 20 min Long synthesis time and catalyst-associated drawbacks are most crucial in silica nanoparticle synthesis. We have addressed both these bottlenecks by replacing the conventional Stober catalyst, ammonium hydroxide, with sodium hydroxide. We have reduced the overall synthesis time from 20 to 1/3 h, ~60-fold decrease, and obtained highly monodispersed nanoparticles with 5-fold higher surface area than Stober particles. We have demonstrated that the developed NPs with ~3-fold higher silane can be used as efficient probes for biosensor applications.