Simple approach to study biomolecule adsorption in polymeric microfluidic channels.

Herein a simple analytical method is presented for the characterization of biomolecule adsorption on cyclo olefin polymer (COP, trade name: Zeonor(®)) substrates which are widely used in microfluidic lab-on-a-chip devices. These Zeonor(®) substrates do not possess native functional groups for specific reactions with biomolecules. Therefore, depending on the application, such substrates must be functionalized by surface chemistry methods to either enhance or suppress biomolecular adsorption. This work demonstrates a microfluidic method for evaluating the adsorption of antibodies and oligonucleotides surfaces. The method uses centrifugal microfluidic flow-through chips and can easily be implemented using common equipment such as a spin coater. The working principle is very simple. The user adds 40 L of the solution containing the sample to the starting side of a microfluidic channel, where it is moved through by centrifugal force. Some molecules are adsorbed in the channel. The sample is then collected at the other end in a small reservoir and the biomolecule concentration is measured. As a pilot application, we characterized the adsorption of goat anti-human IgG and a 20-mer DNA on Zeonor(®), and on three types of functionalized Zeonor: 3-aminopropyltriethoxysilane (APTES) modified surface with mainly positive charge, negatively charged surface with immobilized bovine serum albumin (BSA), and neutral, hydrogel-like film with polyethylene glycol (PEG) characteristics. This simple analytical approach adds to the fundamental understanding of the interaction forces in real, microfluidic systems. This method provides a straightforward and rapid way to screen surface compositions and chemistry, and relate these to their effects on the sensitivity and resistance to non-specific binding of bioassays using them. In an additional set of experiments, the surface area of the channels in this universal microfluidic chip was increased by precision milling of microscale trenches. This modified surface was then coated with APTES and tested for its potential to serve as a unique protein dilution feature.

Improving the sensitivity of immunoassays with PEG-COOH-like film prepared by plasma-based technique.

Herein we report on a preparation and performance of stable, hydrophilic and biocompatible polymeric material suitable for functionalization of disposable substrates used in biosensors. This new material features COOH surface groups cross-linked with ethylene glycol molecules and was prepared in situ on disposable, plastic substrate by high-throughput and environmentally friendly technique called plasma-enhanced chemical vapor deposition (PECVD). The film is grafted to the plasma activated plastic by sequential deposition of tetraethylorthosilicate, forming a bonding layer, and mixed vapors of acrylic acid and diethyleneglycol dimethylether (AA/PEG) that provide the desired functional groups forming a sensing, contact layer. A superior performance of the AA/PEG coating as suitable material for substrates in biomedical devices was demonstrated in a model fluorescence linked immunosorbent assay. The results were compared with other commonly used surface materials prepared by wet chemistry methods. The unique characteristic of the AA/PEG film is that the immunoassay can be executed without the need for a blocking step, typically using albumins, without negative consequences on the bioassay results. In fact, the superior quality of the materials modified with AA/PEG film was highlighted by improving the sensitivity of an immunoassay by two orders of magnitude when compared with substrates prepared by standard surface chemistry methods.

TIRF microscopy as a screening method for non-specific binding on surfaces.

We report a method for studying nanoparticle-biosensor surface interactions based on total internal reflection fluorescence (TIRF) microscopy. We demonstrate that this simple technique allows for high throughput screening of non-specific adsorption (NSA) of nanoparticles on surfaces of different chemical composition. Binding events between fluorescent nanoparticles and functionalized Zeonor® surfaces are observed in real-time, giving a measure of the attractive or repulsive properties of the surface and the kinetics of the interaction. Three types of coatings have been studied: one containing a polymerized aminosilane network with terminal -NH(2) groups, a second film with a high density of -COOH surface groups and the third with sterically restraining branched poly(ethylene)glycol (PEG) functionality. TIRF microscopy revealed that the NSA of nanoparticles with negative surface charge on such modified coatings decreased in the following order -NH(2)>-branched PEG>-COOH. The surface specificity of the technique also allows discrimination of the degree of NSA of the same surface at different pH.

Functionalization of cyclo-olefin polymer substrates by plasma oxidation: stable film containing carboxylic acid groups for capturing biorecognition elements.

Many current designs in biomedical diagnostics devices are based on the use of low cost, disposable, easy-to-fabricate chips made of plastic material, typically a cyclo-olefin polymer (COP). Low autofluorescence properties of such material, among others, make it ideal substrate for fluorescence-based applications. Functionalization of this plastic substrate for biomolecule attachment is therefore of great importance and the quality of films produced on such surface have often a significant influence on the performance of the device. In this communication we discuss the surface chemistry and some other characteristics of hydrophilic films, containing carboxylic acid functional groups, formed by plasma oxidation of COP and also films containing cross-linked, polymerized acryclic acid produced by sequential deposition of tetraorthosilicate and acrylic acid by plasma enhanced chemical vapor deposition (PECVD). Immobilization of labeled, single stranded DNA revealed high binding capacity for both coatings. To our best knowledge, this is the first example of direct immobilization of biomolecules on just plasma oxidized COP. Furthermore, more sophisticated treatment of the oxidized plastic substrate by PECVD with other organic precursors increased the binding capacity by some 40% than that of just plasma oxidized COP. The carboxy functionalized surfaces, due to the negative charge of the carboxy groups, showed very positive trends towards increasing the signal to noise ratio when charged biomolecules such as DNA, are used.

Plasma functionalization of AFM tips for measurement of chemical interactions.

In this paper, a new, fast, reproducible technique for atomic force microscopy (AFM) tips functionalization used for chemical interaction measurements is described. Precisely, the deposition of an aminated precursor is performed through plasma-enhanced chemical vapor deposition (PECVD) in order to create amine functional groups on the AFM tip and cantilever. The advantages of the precursor, aminopropyltriethoxysilane (APTES), were recently demonstrated for amine layer formation through PECVD deposition on polymeric surfaces. We extended this procedure to functionalize AFM probes. Titration force spectroscopy highlights the successful functionalization of AFM tips as well as their stability and use under different environmental conditions.

Functionalization of cycloolefin polymer surfaces by plasma-enhanced chemical vapour deposition: comprehensive characterization and analysis of the contact surface and the bulk of aminosiloxane coatings.

The surface science of bioassay devices is of great importance in the development of modern diagnostic platforms. The quality of surface is one of the most important elements of the device, often governing the background response, hence controlling the sensitivity of an assay. Detailed surface characterization and analysis are imperative for the preparation of reproducible coatings with desired properties. We performed a comprehensive characterization of 3-aminopropyl-triethoxysilane films prepared under two different deposition conditions on COP slides. Two sets of slides were prepared, by exposing them to plasma reaction for 30 seconds (A30 slide) and 4 minutes (A4 slide). While the variations in the deposition conditions seemed very subtle, the use of several powerful analytical tools helped us to reveal some fundamental differences between the studied films in terms of binding capacity, swelling and adhesion. Overall, the A30 films, with a thickness of 5.12 nm, showed up to 40% higher binding capacity and 25% better adhesion than the thicker A4 coatings (28.15 nm). Upon contact with aqueous media, a significant change was observed in terms of surface roughness. The A30 slides outperformed A4 slides, resulting in smoother surface, which is an important parameter for biomolecule immobilisation. The use of the techniques described in this article is aimed to set new standards for the characterization and analysis of the substrate surface of the future diagnostic devices.