Dhanuka P. Wasalathanthri

Attomolar Detection of a Cancer Biomarker Protein in Serum by Surface Plasmon Resonance Using Superparamagnetic Particle Labels

Methods to measure protein biomarkers with ultralow detection limit (DL) and high sensitivity promise to provide valuable tools for early diagnosis of diseases such as cancer, and for monitoring therapy and post-surgical recurrence.[i, ii] Surface plasmon resonance (SPR) utilizing nanoparticle-antibody labels for signal amplification in immunoassays is an emerging approach for detecting proteins in biomedical samples.[iii–x] Herein, we show for the first time that clustering of superparamagnetic labels on SPR surfaces leads to unprecedented sensitivity and ultralow DL for protein biomarkers in serum. Specifically, antibody bioconjugates on 1 µm diameter superparamagnetic particles (MP) used for off-line antigen capture enabled SPR detection of cancer biomarker prostate specific antigen (PSA) in serum at an ultralow DL of 10 fg mL−1 (~300 aM). This approach opens doors for accurate diagnostics based on new protein biomarkers with inherently low concentrations.

Efficient Bioelectronic Actuation of the Natural Catalytic Pathway of Human Metabolic Cytochrome P450s

Cytochrome (cyt) P450s comprise the enzyme superfamily responsible for human oxidative metabolism of a majority of drugs and xenobiotics. Electronic delivery of electrons to cyt P450s could be used to drive the natural catalytic cycle for fundamental investigations, stereo- and regioselective synthesis, and biosensors. We describe herein 30 nm nanometer-thick films on electrodes featuring excess human cyt P450s and cyt P450 reductase (CPR) microsomes that efficiently mimic the natural catalytic pathway for the first time. Redox potentials, electron-transfer rates, CO-binding, and substrate conversion rates confirmed that electrons are delivered from the electrode to CPR, which transfers them to cyt P450. The film system enabled electrochemical probing of the interaction between cyt P450 and CPR for the first time. Agreement of film voltammetry data with theoretical simulations supports a pathway featuring a key equilibrium redox reaction in the natural catalytic pathway between reduced CPR and cyt P450 occurring within a CPR-cyt P450 complex uniquely poised for substrate conversion.

Highly efficient binding of paramagnetic beads bioconjugated with 100,000 or more antibodies to protein-coated surfaces.

We report here the first kinetic characterization of 1 μm diameter superparamagnetic particles (MP) decorated with over 100,000 antibodies binding to protein antigens attached to flat surfaces. Surface plasmon resonance (SPR) was used to show that these antibody-derivatized MPs (MP-Ab(2)) exhibit irreversible binding with 100-fold increased association rates compared to free antibodies. The estimated upper limit for the dissociation constant of MP-Ab(2) from the SPR sensor surface is 5 fM, compared to 3-8 nM for the free antibodies. These results are explained by up to 2000 interactions of MP-Ab(2) with protein-decorated surfaces. Findings are consistent with highly efficient capture of protein antigens in solution by the MP-Ab(2) and explain in part the utility of these beads for ultrasensitive protein detection into the fM and aM range. Aggregation of these particles on the SPR chip, probably due to residual magnetic microdomains in the particles, also contributes to ultrasensitive detection and may also help drive the irreversible binding.

Microfluidic Electrochemical Array for Detection of Reactive Metabolites Formed by Cytochrome P450 Enzymes

A novel, simple, rapid microfluidic array using bioelectronically driven cytochrome P450 enzyme catalysis for reactive metabolite screening is reported for the first time. The device incorporates an eight-electrode screen-printed carbon array coated with thin films of DNA, [Ru(bpy)(2)(PVP)(10)](ClO(4)) {RuPVP}, and rat liver microsomes (RLM) as enzyme sources. Catalysis features electron donation to cyt P450 reductase in the RLMs and subsequent cyt P450 reduction while flowing an oxygenated substrate solution past sensor electrodes. Metabolites react with DNA in the film if they are able, and damaged DNA is detected by catalytic square wave voltammetry (SWV) utilizing the RuPVP polymer. The microfluidic device was tested for a set of common pollutants known to form DNA-reactive metabolites. Logarithmic turnover rates based on SWV responses gave excellent correlation with the rodent liver TD(50) toxicity metric, supporting the utility of the device for toxicity screening. The microfluidic array gave much better S/N and reproducibility than single-electrode sensors based on similar principles.

High-throughput metabolic genotoxicity screening with a fluidic microwell chip and electrochemiluminescence

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 (Ru(II)PVP) 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.

Screening reactive metabolites bioactivated by multiple enzyme pathways using a multiplexed microfluidic system

A multiplexed, microfluidic platform to detect reactive metabolites is described, and its performance is illustrated for compounds metabolized by oxidative and bioconjugation enzymes in multi-enzyme pathways to mimic natural human drug metabolism. The device features four 8-electrode screen printed carbon arrays coated with thin films of DNA, a ruthenium-polyvinylpyridine (RuPVP) catalyst, and multiple enzyme sources including human liver microsomes (HLM), cytochrome P450 (cyt P450) 1B1 supersomes, microsomal epoxide hydrolase (EH), human S9 liver fractions (Hs9) and N-acetyltransferase (NAT). Arrays are arranged in parallel to facilitate multiple compound screening, enabling up to 32 enzyme reactions and measurements in 20-30 min. In the first step of the assay, metabolic reactions are achieved under constant flow of oxygenated reactant solutions by electrode driven natural catalytic cycles of cyt P450s and cofactor-supported bioconjugation enzymes. Reactive metabolites formed in the enzyme reactions can react with DNA. Relative DNA damage is measured in the second assay step using square wave voltammetry (SWV) with RuPVP as catalyst. Studies were done on chemicals known to require metabolic activation to induce genotoxicity, and results reproduced known features of metabolite DNA-reactivity for the test compounds. Metabolism of benzo[a]pyrene (B[a]P) by cyt P450s and epoxide hydrolase showed an enhanced relative DNA damage rate for DNA compared to cyt P450s alone. DNA damage rates for arylamines by pathways featuring both oxidative and conjugative enzymes at pH 7.4 gave better correlation with rodent genotoxicity metric TD(50). Results illustrate the broad utility of the reactive metabolite screening device.