Advancements in microwell array technology: from novel microfabrication strategies to single bead manipulations

Over the years, we witnessed impressive progress in the field of biosensor technology with numerous successful applications for life science research, medical diagnostics and industrial processes. A lot of research effort has been made to achieve better analytical sensitivities with single-molecule resolution as the ultimate goal. Such ultrasensitive detection platforms are extremely valuable for the early detection of low-abundance disease-relevant biomarkers as well as for screening heterogenic sample populations. In particular, bead-based microwell arrays have proven its potential for target detection with single-molecule resolution, by spatially confining individual targets in microwells. The success of ultrasensitive bead-based microwell array technology is exemplified by the commercial applications of Illumina (i.e. BeadArray™) and Quanterix (i.e. SiMoA™) for nucleic acid analysis and ultrasensitive protein measurements, respectively. High-efficiency seeding of magnetic beads is key for the success of these applications and can be enhanced by the creation of hydrophilic-in-hydrophobic (HIH) microwell arrays. However, the fabrication of HIH microwell arrays requires complex and labour-intensive procedures and consequently calls for simpler manufacturing methods. Additionally, current bead-based microwell array systems are restricted solely to target detection, not allowing subsequent manipulation of single beads with captured targets of interest. Although various bead-based trap-and-release systems have already been developed for manipulation of interesting targets at the single-molecule level, they either release all trapped beads at once or in a sequential manner. These platforms thus require an integrated sorting unit, which separates those beads that have captured an interesting target from the rest of the beads. Hence, there is a need for a multipurpose ultrasensitive bead-based trap-and-release system that would enable both detection and manipulation of interesting target molecules. Such platforms would be of special interest for single-cell applications, bacteria screening, drug discovery and antibody therapeutics. Therefore, the aim of this dissertation, is to (i) establish a multipurpose platform for both detection and manipulation of single molecules by integrating an optical manipulation tool (for retrieving single magnetic beads) with the digital microfluidic-based microwell array, and (ii) evaluate an innovative microwell array fabrication method for the swift manufacturing of HIH microwell arrays. The first part of this work focused on the integration of optical tweezers with an electrowetting-on-dielectric-based digital microfluidic microwell array system for the manipulation of single magnetic beads, seeded in a microwell array. Optical manipulation of seeded magnetic beads required their Brownian motion. To this end, the optimal experimental design approach was used for studying and identifying optimal buffer conditions, which generated high fractions of seeded beads with Brownian motion and thus enabled successful manipulation of single magnetic beads. Six different buffer parameters, including bead type, ionic buffer strength, pH, non-ionic surfactant type and concentration, were investigated in this experiment. The analysis of this optimal experimental design indicated different optimal buffer conditions for carboxylated and streptavidin-coated beads, with Tween 40 and Tween 60 as the best non-ionic surfactants. Using these optimized buffer conditions, multiple bead manipulations, including bead retrieval, trapping, transporting and repositioning in another microwell, were successfully demonstrated. In the second part, the potential of a novel hydrophobic off-stoichiometric thiol-ene-epoxy polymer formulation was explored for imprinting HIH microwell arrays, using a stamp-moulding technique. With this method, HIH microwell arrays were created with excellent dimensions and topological features, enabling highly efficient printing and seeding of superparamagnetic beads. Using streptavidin-biotin interaction and β–galactosidase as a reporter enzyme, a digital bioassay was performed. A detection limit of 17.4 attomolar was obtained, demonstrating the potential of the imprinted microwell arrays to perform sensitive digital bioassays. Hence, it can be concluded that the novel hydrophobic Off-stoichiometric Thiol-Ene-Epoxy polymer and the integration of optical tweezers in the electrowetting-on-dielectric-based digital microfluidic microwell array system have a huge potential for the development of new commercial digital ELISA and bead-based trap-and-release system, respectively.status: publishe

Integration of optical tweezers in a digital microfluidic platform for magnetic particle manipulation in a microwell array

Recently, optical tweezers have been used for isolating and manipulating single cells in PDMS microwell arrays showing great potential for cell fusion, fertilization, and cell migration [1, 2]. However, a similar tool has not been developed for single magnetic particles seeded in a Teflon microwell array. The combination of magnetic particles and microwells allows the implementation of digital bioassays for detecting single protein or DNA molecules. This work describes the integration of optical tweezers (Fig. 1A) with a digital microfluidic (DMF) chip bearing a Teflon microwell array (Fig. 1B), to develop a platform for the manipulation of single magnetic particles, functionalized with biomolecules. To obtain this, a double-plated DMF device was used. The DMF top-plate was coated with a thick Teflon layer in which a microwell array was fabricated using a dry lift-off technique [3]. The fabrication process results in a hexagonal array of femtoliter-sized microwells. The microwells are 4.5 μm in diameter and can accommodate only one magnetic particle (Fig. 1B). Figure 1A illustrates the optical tweezer setup used for manipulating single magnetic particles. The infrared laser was focused into an inverted microscope using a beam expansion telescope, beam steering lenses and a high numerical aperture objective. To achieve stable particle trapping, the light intensity gradient force must exceed the light scattering force. Once the focused laser beam was aimed on a particle in a microwell, the particle got attracted to the point of highest light intensity, which is right above the microwell plane. By changing the focus, the magnetic particle could be released in the above droplet (Fig. 2). Using electrowetting-on-dielectric method, the droplet was dragged off the array collecting released particles. It was demonstrated that particles could be retrieved from the microwells only in the presence of certain non-ionic surfactants, which prevent particles from adhering to both Teflon and glass. In order to find a relationship between bead surface properties and retrieval performance differently functionalized particles were tested. Beside the particle retrieval, also the transportation and positioning in other microwells were demonstrated. The following link gives access to a movie illustrating the mechanism of particle retrieval: (https://www.dropbox.com/s/8r3ct2fppc3rkq3/P68_05001.avi). We have combined a microwell array on a DMF chip and optical tweezers technology to obtain a single magnetic particle manipulation tool. This tool shows great potential for bio-receptor screening, digital assays and aptamer selection. The technique also offers the possibility to combine single molecule detection with other on-chip methods (like sequencing), without removing the particles from the chip. [1] X. Wang, X. Gou, S. Chen, X. Yan, and D. Sun, J. Micromechanics Microengineering, vol. 23, no. 7, p. 075006, Jul. 2013. [2] X. Wang, S. Chen, Y. T. Chow, C. Kong, R. a. Li, and D. Sun, RSC Adv., vol. 3, no. 45, p. 23589-23595, 2013. [3] D. Witters, K. Knez, F. Ceyssens, R. Puers, and J. Lammertyn, “Lab Chip, no. 207890, p. 2047-2054, 2013.status: publishe

Surface Nanostructuring of Parylene-C Coatings for Blood Contacting Implants

This paper investigates the effects on the blood compatibility of surface nanostructuring of Parylene-C coating. The proposed technique, based on the consecutive use of O2 and SF6 plasma, alters the surface roughness and enhances the intrinsic hydrophobicity of Parylene-C. The degree of hydrophobicity of the prepared surface can be precisely controlled by opportunely adjusting the plasma exposure times. Static contact angle measurements, performed on treated Parylene-C, showed a maximum contact angle of 158°. The nanostructured Parylene-C retained its hydrophobicity up to 45 days, when stored in a dry environment. Storing the samples in a body-mimicking solution caused the contact angle to progressively decrease. However, at the end of the measurement, the plasma treated surfaces still exhibited a higher hydrophobicity than the untreated counterparts. The proposed treatment improved the performance of the polymer as a water diffusion barrier in a body simulating environment. Modifying the nanotopography of the polymer influences the adsorption of different blood plasma proteins. The adsorption of albumin—a platelet adhesion inhibitor—and of fibrinogen—a platelet adhesion promoter—was studied by fluorescence microscopy. The adsorption capacity increased monotonically with increasing hydrophobicity for both studied proteins. The effect on albumin adsorption was considerably higher than on fibrinogen. Study of the proteins simultaneous adsorption showed that the albumin to fibrinogen adsorbed ratio increases with substrate hydrophobicity, suggesting lower thrombogenicity of the nanostructured surfaces. Animal experiments proved that the treated surfaces did not trigger any blood clot or thrombus formation when directly exposed to the arterial blood flow. The findings above, together with the exceptional mechanical and insulation properties of Parylene-C, support its use for packaging implants chronically exposed to the blood flow

Optical bead manipulation on a digital microfluidic chip for screening biomolecular interactions

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Label-free monitoring of protein-DNA interactions using fluorescent silver nanoclusters

Aptamers are short ssDNA or RNA oligonucleotides that bind targets with high affinity and specificity by folding into defined tertiary structures. Thanks to these properties aptamers represent attractive bio-receptors.[1][2] Traditionally new aptamers are obtained through SELEX, an iterative process in which sequences are selected against target molecules.[3] Generally, aptamer-target interactions are detected in indirect ways relying on labeling of either target or aptamer. However, labeling is not always desirable and label-free detection would be valuable for sensitive detection of binding events. This work explores the use of fluorescent silver nanoclusters (AgNCs) to develop an innovative system for monitoring aptamer-protein interactions. AgNCs are complexes between few Ag atoms and a specific DNA sequence template to stabilize the clusters. In most cases, AgNCs are synthesized either at the 3’ or 5’ end of the template. Our results show that AgNCs can successfully be generated from a template embedded in the middle of a hybridization probe, used for the isothermal amplification of aptamers, called rolling circle amplification (RCA). RCA uses circular oligonucleotide probes to generate long, ssDNA molecules containing periodic repeats of the circular probe.[4][5] Previous works show that overexpression of aptamers by RCA increases target binding efficiency compared to monovalent aptamers.[6] The RCA concatemer combines both the aptamer and the fluorescent AgNC template. Subsequently synthetized AgNCs exhibit strong, robust and tunable fluorescence, eliminating the need for labeling.[7] Importantly, it has been shown that aptamer-AgNCs retain the same specificity and affinity for the cognate protein and that target binding results in a drastic decrease of nanocluster fluorescence.[8] This work explores the integration of an AgNC-aptamer sequence into a circular probe to generate intrinsically fluorescent aptamer concatemers with improved binding efficiency. Possible applications are monitoring target binding during SELEX and label-free ultrasensitive detection of proteins.status: publishe

MAGNETIC PARTICLE RETRIEVAL AND POSITIONING IN A MICROWELL ARRAY BY INTEGRATING OPTICAL TWEEZERS IN A DIGITAL MICROFLUIDIC PLATFORM

© 14CBMS. This manuscript reports the manipulation of single magnetic microparticles confined in a microwell array by integrating optical tweezers in a digital microfluidic platform. In the presence of non-ionic surfactants the magnetic particles are trapped by a tightly focused laser. Once a particle is trapped in the laser beam it can be repositioned in a new microwell or released from the array and transported on the chip for further analysis.status: publishe

Combining DNA origami with aptamers for single molecule counting in microwell arrays

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Ultrasensitive Digital Assay Enables Quantification of Alzheimer Tau Biomarker at sub-femtomolar Concentrations

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