Lidija Malic

Biochip functionalization using electrowetting-on-dielectric digital microfluidics for surface plasmon resonance imaging detection of DNA hybridization

This work reports on a dynamically configurable micro-array surface plasmon resonance biochip platform. The platform comprises a digital electrowetting-on-dielectric (EWOD) microfluidic device tailored to surface plasmon resonance imaging (SPRi). We demonstrate its application for simultaneous immobilization of different DNA probes at the designated detection sites on-chip from sub-microL volume solutions in combination with multichannel label-free real-time detection of subsequent hybridization reactions. Successful on-chip DNA probe dilution and immobilization is also demonstrated using SPRi hybridization detection. Furthermore, active control of the immobilized probe density and orientation is achieved under an applied potential using the electric interface of the EWOD device. For low probe densities, under negative applied potential, the DNA hybridization efficiency is enhanced compared to passive probe immobilization, yielding a two-fold SPR signal increase within only 8min of hybridization. EWOD microfluidic platform coupled with SPRi promises to dramatically increase the speed of detection and quantification of biomolecular interactions while reducing reagent consumption. The proposed system would enable the development of high-throughput, rapid and ultrasensitive detection of biomolecules beyond DNA microarray applications.

Enhanced surface plasmon resonance imaging detection of DNA hybridization on periodic gold nanoposts

We explore periodic gold nanoposts as substrates for the enhanced surface plasmon resonance imaging (SPRi) detection of DNA hybridization. Rigorous coupled-wave analysis was used to model and design the nanopost-based SPRi biosensor. Arrayed gold nanoposts on gold-coated glass substrate, with various widths and periodicity, were fabricated using electron-beam lithography and characterized with scanning electron and atomic force microscopy. A scanning-angle SPRi apparatus was used to conduct the kinetic analysis of DNA hybridization on nanopost-based sensor surface and assess the corresponding SPR signal amplification. Experimental results showed that both the nanostructure size and period influenced the SPR signal enhancement; the optimized 30 nm height, 50 nm size, and 110 nm period nanoposts provided a fivefold SPR signal amplification compared with the plain 50 nm thick gold film used as control.

Designed biointerface using near-infrared quantum dots for ultrasensitive surface plasmon resonance imaging biosensors.

The surface plasmon resonance imaging chip biointerface is fully designed using near-infrared (NIR) quantum dots (QDs) for the enhancement of surface plasmon resonance imaging (SPRi) signals in order to extend their application for medical diagnostics. The measured SPRi detection signal following the QD binding to the surface was amplified 25-fold for a 1 nM concentration of single-stranded DNA (ssDNA) and 50-fold for a 1 μg/mL concentration of prostate-specific antigen (PSA), a cancer biomarker, thus substantiating their wide potential to study interactions of a diverse set of small biomolecules. This significant enhancement is attributed to the QDs mass-loading effect and spontaneous emission coupling with propagating surface plasmons, which allowed the SPRi limit of detection to be reduced to 100 fM and 100 pg/mL for ssDNA and PSA, respectively. Furthermore, this study illustrates the potential of SPRi to be easily integrated with fluorescent imaging for advanced correlative surface-interaction analysis.

Nanostructured digital microfluidics for enhanced surface plasmon resonance imaging

The advances in genomics and proteomics have unveiled an exhaustive catalogue of biomarkers that can potentially be used as diagnostic and prognostic indicators of genetic and infectious diseases. Current thrust in biosensor development is towards rapid, real-time, label-free and highly sensitive detection of the indicative biomarkers. While surface plasmon resonance imaging (SPRi) biosensors could potentially be the best suited candidate for biomarker-based diagnosis, important milestones need to be reached. Commercially available SPRi instrumentation is currently limited by the flow-cell technology to serial-sample processing and has limited sensitivity for the detection of markers present at low concentration. In this paper, we have implemented an approach to enhance sample handling and increase the sensitivity of the SPRi detection technique. We have developed a digital microfluidic platform with an integrated nanostructured biosensor interface that allows for rapid, ultra-low volume, sensitive, and automated on-chip SPRi detection of DNA hybridization reactions. Through the exploitation of electromagnetic properties of nanofabricated periodic gold nanoposts, SPRi signal was increased by 200% with the estimated limit of detection of 500 pM (90 attomoles). Using the versatile fluidic manipulation provided by the digital microfluidics, rapid and parallel target identification was achieved on multiple array elements within 1 min using 180 nL sample volume. By delivering multiple target analytes in individually addressable low volume droplets, without external pumps and fluidic interconnects, the overall assay time, cost and complexity was reduced. The proposed platform allows extreme versatility in the manipulation of precious low volume samples which makes this technology very suitable for diagnostic applications.

Nanoporous twinned PtPd with highly catalytic activity and stability

Growing needs for highly efficient energy storage devices have prompted increasing research efforts in energy-efficient and sustainable materials. In this context, nanoporous noble metals have been studied extensively because of their extraordinary properties. However, existing electrochemical/chemical dealloying approaches for their synthesis largely lack the ability to optimize their structure/function relationships. To overcome this limitation, we developed a thermal-decomposition strategy for the synthesis of component-controllable nanoporous PtPd alloys composed of ∼2 nm sawtooth-like ligaments induced by a high density of twinning boundaries (boundary spacing ∼ 1 nm). Such twinned and ultrathin ligaments exhibit large curvatures between concave and convex regions, associated with abundant low-coordination surface atomic steps and kinks. These low-coordination atoms are sites of high catalytic activity, as confirmed by theoretical simulations. The optimized Pt25Pd75 sample exhibits the best catalytic performance among all the currently reported catalysts, and has a mass activity of 1110 mA mg−1Pt−1 and high stability for the electro-oxidation of methanol.

Current State of Intellectual Property in Microfluidic Nucleic Acid Analysis

The development of novel fabrication methods, materials and surface chemistries to implement nucleic acid analysis brings reduced cost, reduced reagent consumption, increased analysis efficiency, portability, ease of use and reliability to todays genomic approach. This trend, as evident by the exponential growth in the number of patent applications, granted patents and commercialized systems, is motivated by the promise for significant breakthroughs and benefits of nucleic acid analysis to drug discovery and point-of-care diagnosis. This review paper aims at identifying the enabling technologies and key patents in microfluidics for nucleic acid analysis. In particular, it seeks to identify granted and pending patents for cell sorting and lysis, nucleic acid extraction and purification, followed by nucleic acid amplification, separation and detection. Additionally, it presents an overview of the current intellectual property environment and seeks to identify trends for the future development. Much of this development is geared increasingly toward fully integrated systems. The convergence of technology and interdisciplinary interests is expected to foster further breakthroughs and commercialization.

All-thermoplastic nanoplasmonic microfluidic device for transmission SPR biosensing

Early and accurate disease diagnosis still remains a major challenge in clinical settings. Biomarkers could potentially provide useful tools for the detection and monitoring of disease progression, treatment safety and efficacy. Recent years have witnessed prodigious advancement in biosensor development with research directed towards rapid, real-time, label-free and sensitive biomarker detection. Among emerging techniques, nanoplasmonic biosensors pose tremendous potential to accelerate clinical diagnosis with real-time multiplexed analysis, rapid and miniaturized assays, low sample consumption and high sensitivity. In order to translate these technologies from the proof-of-principle concept level to point of care clinical diagnosis, integrated, portable devices having small footprint cartridges that house low-cost disposable consumables are sought. Towards this goal, we developed an all-polymeric nanoplasmonic microfluidic (NMF) transmission surface plasmon resonance (SPR) biosensor. The device was fabricated in thermoplastics using a simple, single step and cost-effective hot embossing technique amenable to mass production. The novel 3D hierarchical mold fabrication process enabled monolithic integration of blazed nanogratings within the detection chambers of a multichannel microfluidic system. Consequently, a single hard thermoplastic bottom substrate comprising plasmonic and fluidic features allowed integration of active fluidic elements, such as pneumatic valves, in the top soft thermoplastic cover, increasing device functionality. A simple and compact transmission-based optical setup was employed with multiplexed end-point or dual-channel kinetic detection capability which did not require stringent angular accuracy. The sensitivity, specificity and reproducibility of the transmission SPR biosensor was demonstrated through label-free immunodetection of soluble cell-surface glycoprotein sCD44 at clinically relevant picomolar to nanomolar concentrations.

Nanoimprinted plastic substrates for enhanced surface plasmon resonance imaging detection

Periodic nanostructures fabricated by Nanoimprint Litography (NIL) in low-cost plastic substrates and coated with thin gold film were explored for enhanced surface plasmon resonance imaging (SPRi) detection. Rigorous coupled-wave analysis was used to model the SPRi response of these nanostructured surfaces. Two-dimensional nanogratings and nanogrooves were fabricated on Zeonor 1060R(TM) by NIL and followed by metal deposition. The detection of refractive index changes in the dielectric layer due to bulk medium change, DNA immobilization and DNA hybridization events were monitored using SPRi to assess the corresponding signal amplification. The results indicate target-dependent sensitivity enhancement which is maximized for the detection of biomolecular binding events. The 500 nm period nanogrooves provided a 4 times SPR signal amplification compared to the conventional uniform gold film on SF-11 glass for DNA hybridization detection. Our work demonstrates that the use of nanoimprinted plastic substrates provides a low-cost solution for the SPR-based detection with sensitivity that meets the requirements in practical diagnostic applications.

Advanced EWOD-based digital microfluidic system for multiplexed analysis of biomolecular interactions

This paper presents a low-cost technique for the fabrication of complex electrowetting-on-dielectric (EWOD) digital microfluidic devices. Using this original technology, we have developed devices in which 560 electrodes are used to mix and split nl-size liquid droplets and transport them to 100 analysis spots patterned on a disposable plastic top plate. We demonstrate the multiplexing capability of the developed devices by creating on-chip arrays of droplets with various concentration gradients. Finally, automated biomolecular immobilization and hybridization assays are performed in nl-size droplets under numerous conditions simultaneously with only a limited number of stock solutions.