Biocompatible bonding of a rigid off-stoichiometry thiol-ene-epoxy polymer microfluidic cartridge to a biofunctionalized silicon biosensor

Attachment of biorecognition molecules prior to microfluidic packaging is advantageous for many silicon biosensor-based lab-on-a-chip (LOC) devices. This necessitates biocompatible bonding of the microfluidic cartridge, which, due to thermal or chemical incompatibility, excludes standard microfabrication bonding techniques. Here, we demonstrate a novel processing approach for a commercially available, two-step curable polymer to obtain biocompatible ultraviolet initiated (UVA)-bonding of polymer microfluidics to silicon biosensors. Biocompatibility is assessed by UVA-bonding to antibody-functionalized ring resonator sensors and performing antigen capture assays while optically monitoring the sensor response. The assessments indicate normal biological function of the antibodies after UVA-bonding with selective binding to the target antigen. The bonding strength between polymer and silicon chips (non-biofunctionalized and biofunctionalized) is determined in terms of static liquid pressure. Polymer microfluidic cartridges are stored for more than 18 weeks between cartridge molding and cartridge-to-silicon bonding. All bonded devices withstand more than 2500 mbar pressure, far exceeding the typical requirements for LOC applications, while they may also be de-bonded after use. We suggest that these characteristics arise from bonding mainly through intermolecular forces, with a large extent of hydrogen bonds. Dimensional fidelity assessed by microscopy imaging shows less than 2% shrinkage through the molding process and the water contact angle is approximately 80°. As there is generally little absorption of UVA light (365 nm) in proteins and nucleic acids, this UVA-bonding procedure should be applicable for packaging a wide variety of biosensors into lab-on-a-chip systems.acceptedVersio

Microscale tools for the development of bacterial microarrays

Heterogeneity within bacterial populations is a phenomenon that has gained much interest over the last decades. It has been shown that even isogeneous colonies under homogeneous conditions can display different phenotypes. This heterogenous gene expression in bacteria is considered to be an evolutionary developed trait that increases the chance of survival under changing environmental conditions. This also impacts human health as some phenotypic traits can enable bacteria to survive antibiotic treatment, resulting in reoccurring bacterial infections. There is understandably much interest in uncovering the underlying mechanisms of such phenotypic differences, both for optimized medical treatments and to improve our understanding of the behavior of bacterial populations. Standard methods utilized in microbiology are however based on average measurements, and hereby inherently masking the existence of small subpopulations and other rare events. With the emergence of techniques capable of large scale single cell measurements, e.g. flow cytometry, much focus has been put on the understanding of heterogeneity of bacterial populations. There is however a need for single cell measurements that provide time resolution in order to study the dynamics of such phenomena. Such time resolution can be obtained through time laps imaging of bacteria. Large scale single cell measurements could however benefit from an ordered attachment of bacteria onto a substrate. In this thesis I present methods for fabrication of bacterial microarrays, focusing on utilizing methods and chemicals that are applicable in standard biological laboratories. The presented method is based on micro contact printed patterns of chemicals on glass substrates for the selective adhesion of bacteria. Such arrays were utilized to inspect the heterogeneity in expression of green fluorescent protein from two different plasmids carried by the bacteria. The results were comparable to results obtained based on measurements of the same system preformed on a flow cytometer. The surface patterning technique presented was also adapted for the selective adhesion of alginate microgels onto glass substrates. Encapsulation of cells in such alginate microgels allowed for inspection of three dimensional culture growth and the possibility of selective removal of single microgels utilizing a micropipette controlled by a micromanipulator

The Design of Simple Bacterial Microarrays: Development towards Immobilizing Single Living Bacteria on Predefined Micro-Sized Spots on Patterned Surfaces.

In this paper we demonstrate a procedure for preparing bacterial arrays that is fast, easy, and applicable in a standard molecular biology laboratory. Microcontact printing is used to deposit chemicals promoting bacterial adherence in predefined positions on glass surfaces coated with polymers known for their resistance to bacterial adhesion. Highly ordered arrays of immobilized bacteria were obtained using microcontact printed islands of polydopamine (PD) on glass surfaces coated with the antiadhesive polymer polyethylene glycol (PEG). On such PEG-coated glass surfaces, bacteria were attached to 97 to 100% of the PD islands, 21 to 62% of which were occupied by a single bacterium. A viability test revealed that 99% of the bacteria were alive following immobilization onto patterned surfaces. Time series imaging of bacteria on such arrays revealed that the attached bacteria both divided and expressed green fluorescent protein, both of which indicates that this method of patterning of bacteria is a suitable method for single-cell analysis

Right: Fluorescence micrograph of quantum dots deposited on a cleaned glass coverslip using μCP with PDMS stamps.

<p>Such images were used to study the reproducibility of the obtained patterns. Left: Distributions of observed diameters of the nine largest stamped islands compared to the mask hole diameters (blue triangles and corresponding blue linear regression line). Island diameters calculated from the area of each island as determined based on the ImageJ software and fluorescence micrographs of quantum dots. The red triangle indicate the most probable island diameter <i>d</i>_<i>m</i> and the red line is the linear regression obtained based on <i>d</i>_<i>m</i> obtained for the eight largest stamped islands.</p

Time laps images of <i>P. putida</i> KT2440 immobilized on PD islands printed on a PEGylated glass surface.

<p>The images are obtained for surfaces covered with medium between 0 and 160 minutes after changing the medium from LB to LB containing the inducer MB. MB induces the expression of GFP in the bacteria. The images are overlays of transmission light images and fluorescence images—both obtained using a Leica SP5 with a 20 × objective (N.A. = 0.7).</p

Quantitative analysis of the number of bacteria immobilized onto each adhesive PD island of bacterial microarrays prepared on glass surfaces coated with PEG.

<p>The arrays were prepared using μCP with PDMS stamps obtained using a photolithography mask with <i>d</i>_<i>h</i> equal to 3.5 μm (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128162#pone.0128162.g001" target="_blank">Fig 1d</a>). <i>N</i>_<i>b</i> ≥ 1: one or more bacteria per island. <i>N</i>_<i>b</i> = 1: one bacterium per island.</p><p>Quantitative analysis of the number of bacteria immobilized onto each adhesive PD island of bacterial microarrays prepared on glass surfaces coated with PEG.</p

Tapping mode AFM height topographs of PD printed on PEGylated glass.

<p>Tapping mode AFM height topographs of PD printed on PEGylated glass.</p

Images of glass surfaces and glass surfaces precoated with chemicals reducing bacterial adhesion, patterned with chemicals promoting bacterial adhesion, immersed in a solution containing bacteria and finally rinsed and covered with LB.

<p>Results obtained for the three chemicals potentially reducing bacterial adhesion (BSA, PVA or PEG) are shown. The substrates are patterned with one of three chemicals promoting bacterial adhesion (PLL, PEI or PD) using μCP with a PDMS stamp with 5 μm lines (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128162#pone.0128162.g001" target="_blank">Fig 1d</a>) and immersed in a solution containing bacteria. All scalebars are 10 μm. The combination of chemicals investigated in each experiment is indicated on the figure. The surfaces were rinsed in MilliQ water after the incubation with bacteria (<i>P. putida</i> KT2440) in order to remove weakly adhering bacteria. During imaging the surfaces were covered with LB in order to minimize stress induced in the attached bacteria. The images are obtained by using transmission light microscopy, and were captured on a Leica TCS SP5 with a 40 × objective (water, N.A. = 1.2).</p

Impact of Silanization Parameters and Antibody Immobilization Strategy on Binding Capacity of Photonic Ring Resonators

Ring resonator-based biosensors have found widespread application as the transducing principle in “lab-on-a-chip” platforms due to their sensitivity, small size and support for multiplexed sensing. Their sensitivity is, however, not inherently selective towards biomarkers, and surface functionalization of the sensors is key in transforming the sensitivity to be specific for a particular biomarker. There is currently no consensus on process parameters for optimized functionalization of these sensors. Moreover, the procedures are typically optimized on flat silicon oxide substrates as test systems prior to applying the procedure to the actual sensor. Here we present what is, to our knowledge, the first comparison of optimization of silanization on flat silicon oxide substrates to results of protein capture on sensors where all parameters of two conjugation protocols are tested on both platforms. The conjugation protocols differed in the chosen silanization solvents and protein immobilization strategy. The data show that selection of acetic acid as the solvent in the silanization step generally yields a higher protein binding capacity for C-reactive protein (CRP) onto anti-CRP functionalized ring resonator sensors than using ethanol as the solvent. Furthermore, using the BS3 linker resulted in more consistent protein binding capacity across the silanization parameters tested. Overall, the data indicate that selection of parameters in the silanization and immobilization protocols harbor potential for improved biosensor binding capacity and should therefore be included as an essential part of the biosensor development process

(a): The patterns on the photolithography masks used to produce PDMS stamps.

<p>The first pattern (left) consisted of 13 circular holes of diameter increasing from 0.8 μm to 4.4 μm on an opaque background. The mask contained four quadrants, each characterized by a vertical separation distance <i>d</i>₁ of 3, 4, 6 or 8 μm between the circular holes and a fixed horizontal distance <i>d</i>₂ between the center of each hole of 7.4, 8.4, 10.4 or 12.4 μm. The pattern on the second photolithography mask (right) consisted of circular holes with a diameter <i>d</i>_<i>h</i> of 3.5 μm with a separation distance <i>d</i>₃ between the circular holes equal to either 10 or 15 μm. (b), (c) and (d): SEM micrographs of gold coated PDMS stamps intended for patterning of glass surfaces by μCP. The stamps shown in (b) and (c) are produced using the photolithography masks schematically illustrated in 1(a). The stamp depicted in (d) was obtained using a photolithography mask with slits of width 5 μm interspaced by 5 μm.</p