Ram Prasad Gandhiraman

High efficiency amine functionalization of cycloolefin polymer surfaces for biodiagnostics

Point-of-care (POC) diagnostics implementing microfluidic technology on single use disposable plastic chips has potential applications in personalized medicine, clinical diagnostics and global health. However, the challenges in commercializing POC devices must be addressed. Immobilization of biomolecules to plastic chips through appropriate surface functionalization is a key issue for the fabrication of new generation biomedical diagnostic devices. The most important requirements for a practicable surface functionalization process are speed, control and reliability. Plasma-based methods can meet these criteria. A single step, solventless, ecofriendly and high throughput nature of plasma processing makes them highly attractive. Here we demonstrate the efficient surface functionalization of a next-generation biosensor material, a chemically inert cycloolefin polymer (COP). The plasma formation of a surface-bound aminated siloxane network from mixed aminopropyltriethoxysilane and ethylenediamine precursors allowed us to form a well-adherent film with an exceptionally high degree of amine functionalization. We deduce that the siloxane was the critical component for radical insertion into the COP and for building a stable network to support the reactive amine functionalities. We present a full physical and chemical characterization of the films, including a detailed study of their swelling in water, using an array of surface analytical techniques: X-ray photoelectron spectroscopy, X-ray reflectivity, reflection infra-red spectroscopy, atomic force microscopy (AFM) and fluorophore binding reactions. We demonstrate an original approach for qualitatively analyzing the distribution of amine functionalities by counting surface-bound functionalized silica nanoparticles in the AFM. The relative contributions from covalent (specific) and non-covalent (non-specific) reaction chemistry assessed using 3′-fluorescein-labeled ssDNA attachment showed that the non-specific binding could be reduced significantly according to the particular feed gas mixture used to prepare the coating. A reaction mechanism has been proposed for the deposition of amine functionalities on COP plastic and also for enhancing the amine functionalities that affect the non-specific binding significantly.

Reactive amine surfaces for biosensor applications, prepared by plasma-enhanced chemical vapour modification of polyolefin materials

Here we have demonstrated a solventless plasma-based process that integrates low-cost, high throughput, high reproducibility and ecofriendly process for the functionalization of the next-generation point-of-care device platforms. Amine functionalities were deposited by plasma-enhanced chemical vapour deposition (PECVD) using a new precursor. The influence of the plasma RF power and the deposition time on surfacial properties, as well as their effect on the reactivity and content of amino groups was investigated. The key process determinants were to have a sufficient power in the plasma to activate and partially fragment the monomer but not too much as to lose the reactive amine functionality, and sufficient deposition time to develop a reactive layer but not to consume or erode the amine reactivity. An immunoassay performed using human immunoglobulin (IgG) as a model analyte showed an improvement of the detection limit by two orders of magnitude beyond that obtained using devices activated by liquid-phase reaction.

Deposition of chemically reactive and repellent sites on biosensor chips for reduced non-specific binding.

The performances of new polymeric materials with excellent optical properties and good machinability have led the biomedical diagnostics industry to develop cheap disposable biosensor platforms appropriate for point of care applications. Zeonor, a type of cycloolefin polymer (COP), is one such polymer that presents an excellent platform for biosensor chips. These polymer substrates have to be modified to have suitable physico-chemical properties for immobilizing proteins. In this work, we have demonstrated the amine functionalization of COP substrates, by plasma enhanced chemical vapour deposition (PECVD), through codeposition of ethylene diamine and 3-aminopropyltriethoxysilane precursors, for building chemistries on the plastic chip. The elemental composition, adhesion, ageing and reactivity of the plasma polymerized film were examined. The Si-O functionality present in amino silane contributed for a good interfacial adhesion of the coating to COP substrates and also acted as a network building layer for plasma polymerization. Wet chemical modification was then carried out on the amine functionalized chips to create chemically reactive isothiocyanate sites and protein repellent fluorinated sites on the same chip. The density of the reactive and repellent sites was altered by choosing appropriate mixtures of homofunctional phenyldiisothiocyanate (PDITC), pentafluoroisothiocyanate (5FITC) and phenylisothiocyanate (PITC) compounds. By tailoring the density of reactive binding sites and protein repellent sites, the non-specific binding of ssDNA has been decreased to a significant extent.

Plasma Surface Modification of Cyclo-olefin Polymers and Its Application to Lateral Flow Bioassays

The modification of cyclo-olefin polymer Zeonor by plasma-enhanced chemical vapor deposition to form a silica-like surface and evaluation of its application for lateral flow bioassays applications are discussed in this study. The SiOx layer was extensively characterized using contact angle measurements, atomic force microscopy, and Fourier transform infrared spectroscopy in attenuated total internal reflectance mode where the presence of a uniform SiOx film was clearly identified. The SiOx modification resulted in a surface with enhanced wettability and excellent fluidic properties when combined with a hot-embossed micropillar capillary fill-based substrate. The SiOx surface also had the ability to accelerate the clotting of human plasma, which may have application in certain types of blood coagulation assays.

Total internal reflection ellipsometry as a label-free assessment method for optimization of the reactive surface of bioassay devices based on a functionalized cycloolefin polymer

AbstractWe report a label-free optical detection technique, called total internal reflection ellipsometry (TIRE), which can be applied to study the interactions between biomolecules and a functionalized polymer surface. Zeonor (ZR), a cycloolefin polymer with low autofluorescence, high optical transmittance and excellent chemical resistance, is a highly suitable material for optical biosensor platforms owing to the ease of fabrication. It can also be modified with a range of reactive chemical groups for surface functionalization. We demonstrate the applications of TIRE in monitoring DNA hybridization assays and human chorionic gonadotrophin sandwich immunoassays on the ZR surface functionalized with carboxyl groups. The Ψ and Δ spectra obtained after the binding of each layer of analyte have been fitted to a four-layer ellipsometric model to quantitatively determine the amount of analytes bound specifically to the functionalized ZR surface. Our proposed TIRE technique with its very low analyte consumption and its microfluidic array format could be a useful tool for evaluating several crucial parameters in immunoassays, DNA interactions, adsorption of biomolecules to solid surfaces, or assessment of the reactivity of a functionalized polymer surface towards a specific analyte. Figure(a) Total internal reflection ellipsometry (TIRE) experimental setup (b) Typical Ψ and Δ spectra of the sensing substrate measured in phosphate-buffered saline (PBS)

Evaluation of different nonspecific binding blocking agents deposited inside poly(methyl methacrylate) microfluidic flow-cells.

Poly(methyl methacrylate) (PMMA) flow-cells containing microwells were deposited with different nonspecific binding blocking agents, namely, bovine serum albumin (BSA), cationic lipid (DOTAP:DOPE) and diethylene glycol dimethyl ether (DEGDME). Water contact angle (WCA) and atomic force microscope (AFM) measurements were carried out to confirm the successful depositions of BSA, DOTAP, and DEGDME onto the PMMA surfaces. Fluorescent intensity measurements were performed to evaluate the degree of nonspecific adsorption of Cy5-labeled anti-IgG proteins onto plain and oxygen plasma-treated (PT) PMMA flow-cells as well as PMMA flow-cells deposited with different above-mentioned blocking agents. We then employed a label-free detection method called total internal reflection ellipsometry (TIRE) to evaluate the stability of the deposited blocking agents inside the PMMA flow-cells. It was found that, while DOTAP:DOPE was the best agent for blocking the nonspecific adsorption, it could be removed from the PMMA surfaces of the flow-cells upon rinsing with phosphate buffered saline (PBS) and later deposited back onto the Au-coated glass sensing substrate of the TIRE. The removal of the blocking agents from PMMA surfaces and their deposition onto the sensing substrate were further manifested by measuring the kinetics and the amount of adsorbed anti-α-hCG proteins. Overall, the dry DEGDME coating by plasma-enhanced chemical vapor deposition (PECVD) showed very good blocking and excellent stability for subsequent assay inside the microwells. Our results could be useful when one considers what blocking agents should be used for PMMA-based microfluidic immunosensor or biosensor devices by looking at both the blocking efficiency and the stability of the blocking agent.

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.

Multi-Layered Plasma-Polymerized Chips for SPR-Based Detection

The surface functionalization of a noble metal is crucial in a surface plasmon resonance-based biomolecular detection system because the interfacial coating must retain the activity of immobilized biomolecules while enhancing the optimal loading. We present here a one-step, room-temperature, high-speed, gas-phase plasma polymerization process for functionalizing gold substrates using siloxane as an adhesion layer and acrylic acid as a functional layer. Siloxane- and thiol-based coatings were compared for their performance as adhesion and the interfacial layer for subsequent functionalization. An in situ sequential deposition of siloxane and acrylic acid resulted in a 7-fold increase in carboxylic functionality surfacial content compared to films deposited with thiol-containing precursors. Grading of the layer composition achieved as a consequence of ion-induced mixing on the surface coating under the application of the plasma is confirmed through secondary ion mass spectroscopic studies. DNA hybridization assays were demonstrated on gold/glass substrates using surface plasmon enhanced ellipsometry and the applicability of this coating for protein immunoassays were demonstrated with plasma functionalized gold/plastic substrates in Biacore 3000 SPR instrument.

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.