Microfluidic systems have been highly evolving with the simultaneous development of polymer materials. Polymer technology has been key in therealization and definition of the so-called Lab-on-a-Chip \(LoC) concept. The current high impact of LoC systems is partly due to the application ofpolymers such as polycarbonate \(PC), poly \(methyl methacrylate) \(PMMA), SU-8 and poly\(dimethylsiloxane) \(PDMS), which have made them more versatilewhile in turn have enabled reducing their fabrication cost and time. PDMS \(Figure 1a) is a cheap material that polymerizes at low temperatures1. It is optically transparent in a very wide wavelength range, from ultra-violet \(UV) to the Near-Infrared \(NIR)2. This lastproperty makes the material compatible with many optical detection methods. It is also compatible with biological studies, since it is non-permeable towater, non toxic and permeable to gases. PDMS is an elastomer with a 2.5 MPa Young modulus when prepared with a 10:1 ratio of a base:curing agent3. Cast molding of the as-prepared PDMS provides a rapid fabrication of microsystems with resolution down to 0.1 µm4. Theresulting systems can easily be sealed to many different substrates3. When LoCs are fabricated with PDMS, low-cost systems can be obtainedwith the potential of being highly sensitive. However, this polymer has a disadvantage: biomolecules and other macromolecules easily adsorbnon-specifically to it, thus hindering its application for chemical sensing. This disadvantage can easily be turned into anadvantage as it can easily be modified in order to avoid that process or, by contrast, to selectively immobilize different molecules2. Thesesurface modification processes are usually needed for the application of PDMS-based microsystems to \(bio)chemical analysis. The aims of themodification are diverse and include from the minimization of the biomolecular adsorption, to the increase of the hydrophilic/hydrophobic character ofthe surface. Some processes are directed to bind a biologically active molecule that changes the lubricity of the surface5 or provides thematerial with the capacity to give a selective answer to a specific target analyte by binding antibodies6 or enzymes7.
Biofunctionalization of PDMS surfaces can be carried out following two different strategies: physical adsorption and covalent modification. The firstone is very simple but, due to the weak interactions between the adsorbed molecules and the surface, the modifications are instable both thermally andmechanically. Also, solvolytic processes can also occur. Covalent modification can overcome these problems and provide more stable modifications8. It is carried out by the initial introduction of hydroxyl groups \(–OH) on the PDMS surface, which can further be modified by asilanization process. These hydroxyl groups react with silane molecules to form covalent Si-O-Si \(siloxane) bonds. Different functional groups, towhich the biomolecules can be covalently attached, are introduced on the surface depending on the chosen silane9. In this context, PDMSsurfaces have been treated with oxygen plasma10 or UV/ozone11, in order to make the surface hydrophilic by replacing the surfacemethyl groups, bound to the Si atom within the PDMS structure, by silanol groups \(Si-OH). These new groups tend to chemically interact with otherfunctional groups, allowing to selectively modify the surface. Silanol groups can be useful as an initial step in the PDMS surface modification forcovalently binding enzymes, as Yasukawa et al. did7. They immobilized glucose oxidase on a PDMS layer after a hydrophilization stepusing a plasma process and further silanization, with the aim of fabricating a glucose sensor. Sandison et al. made also use of a plasma andsilanization process to immobilize antibodies on a PDMS column for protein purification applications12. But this process also has somedrawbacks: the modification is temporal because the plasma oxidized surface progressively recovers its hydrophobicity. It also requires specialinstrumentation and cannot be applied in the microfluidic channels of LoCs13. This means that alternative processes must be found toselectively modify PDMS surfaces, which were easy to implement and could be applied in channels embedded in PDMS matrices.
The previously mentioned UV/ozone treatment could be an alternative process. The modification consists in firstly generating ozone from molecularoxygen by 185 nm wavelength light exposition and then photodissociating it to atomic oxygen under 254 nm wavelength light exposure. This oxygenabstracts hydrogen from the backbone of PDMS and silanol \(Si–OH) structures are formed on it, becoming a hydrophilic surface14. Thistreatment is slower than a plasma activation process15 but it facilitates a much deeper modification without cracking or mechanicalweakening side-effects11. This fact enables its application to the microchannels. But, as pointed out before, the process is reversible, andPDMS surface eventually recovers its hydrophobicity after exposure to air for a few hours.
Chemical Vapour Deposition \(CVD) can also be used to create polymer coatings on PDMS microchannels, as Chen and Lahann did for the eventual depositionof poly\(4-benzoyl-p-xylylene-co-p-xylylene) films. A light reactive coating film of carbonyl groups was obtained, which was exposed to UV light inorder to generate the free radicals that could react with poly\(ethylene oxide) \(PEO) and create PEO-functionalized regions that avoided the adsorptionof fibrinogen16.
Silanol groups can also be obtained on PDMS surfaces by using sol–gel methods. Silica nanoparticles can be created in a PDMS piece by mixing itin a tetraethyl orthosilicate \(TEOS) sol–gel precursor and then incubating it in an ethylamine catalyzing solution and heating it17.Glasslike layers can also be formed on a PDMS surface by applying the same sol–gel technique with transition metal sol-gel precursors18. However, these sol-gel methods are time consuming and therefore the production costs increase.
An acidic solution containing hydrogen peroxide \(H2O2) can also be pumped inside the microchannels, which oxidizes the PDMSsurface and creates silanol groups13.The process should be carefully controlled since an excess of acidity could lead to a loss of opticaltransparency of the PDMS.
Physical adsorption methods can also be applied for PDMS microchannel modification. These methods are applied to suppress electroosmotic flow incapillary electrophoresis and to avoid nonspecific binding of proteins. The hydrophobic parts of molecules can be physisorbed onto the PDMS surfacewhile the hydrophilic parts keep exposed to the buffer, thus changing the surface properties of the PDMS. A coating process of polymers that containhydrophobic and hydrophilic parts can be achieved by simply incubating the surface with the aqueous coating solution19. The so-called Layerby Layer \(LBL) technique can also be carried out by electrostatic adsorption of positively and negatively charged alternating layers20.
Here, the details of the protocol used in a previous work21 for different liquid-based surface chemical biofunctionalization methods areprovided. The developed methods can easily be performed in standard chemical and biological laboratories avoiding the need of special instrumentation.Both physical adsorption and covalent modification methods are analyzed. On one hand, physical adsorption of two different polymers containing hydroxylgroups, such as polyethylene glycol \(PEG) or polyvinyl alcohol \(PVA) \(figure 1b) enable the further silanization of the surface for the introduction ofchemical functional groups and the eventual covalent immobilization of the bioreceptor. In some applications, as Yu et al did22, theaim of the PVA immobilization was to avoid the non-specific binding of proteins. Other groups used PEG instead of PVA, since it offers the sameadvantage23,24. In the present application, the objective is totally different. These polymers are used as anchoring points for furthersilanization and final protein receptor immobilization. On the other hand, a covalent modification approach was tested based on the chemical oxidationof the PDMS surface that generates silanol groups \(figure 1c) onto which a silanization process and further immobilization of the protein receptor arecarried out, as above. This chemical oxidation protocol was already described by Sui et al. for creating hydroxyl groups that could be used asanchoring points for the immobilization of other molecules13. A deep structural characterization of the resulting modified surfaces iscarried out. The analytical performance and the stability of the modified surfaces following the different methods are also tested using a PDMS-basedphotonic LoC \(PhLoC) microsystem consisting of a hollow Abbe prism transducer configuration25.
Modification of the PDMS surfaces
As it can be seen in Figure 2, the proposed three approaches for PDMS surface modification are based on the introduction of hydroxyl \(-OH) groups andfurther silanization. PDMS surfaces were cleaned with ethanol and deionized water \(DI H2O). For the modification shown in Figure 2A, thePDMS surfaces were incubated in a PEG solution and left to adsorb. For the modification in Figure 2B, they were incubated in a PVA solution and left toadsorb. The backbone of these two polymers is able to physisorb from aqueous solutions to hydrophobic surfaces26. The third approach wascarried out by a chemical oxidation process with an acidic solution containing DI H2O, HCl and H2O213\(Figure 2C). After each of these steps, the surfaces were rinsed with DI H2O and dried.
For the previously mentioned silanization process, the modified PDMS systems were incubated in 11-triethoxysilyl undecanal \(TESU) and triethylamine\(TEA) containing ethanol solutions. Then the surfaces were thoroughly rinsed with ethanol and dried. The TEA induces a highly nucleophilic oxygen inthe –OH group that readily interacts with the chosen silane27 having its ethoxy groups previously hydrolyzed. In this way, the silanemolecules covalently bound to the surface \(Figure 3).
Characterization of the PDMS surfaces
For the characterization process, flat PDMS surfaces were modified. The techniques used for this characterization were contact angle measurements, XPSand AFM.
Contact angle measurements were carried out with the sessile drop method. Images with a high contrast should be obtained with the camera of the anglemeter, with as less light reflections as possible in the drop. If these conditions are hold, the software is able to automatically detect the shape ofthe drop and measure the contact angle.
XPS analysis was carried out on an Axis Ultra-DLD spectrometer, using a monochromatized Al Kαsource \(1486.6 eV). Signals were deconvoluted with the software provided by the manufacturer, using a weighted sum of Lorentzian and Gaussian componentcurves after background subtraction. The binding energies were referenced to the internal standard C 1s \(284.9 eV).
Atomic force microscopy topographic and phase images of the modified surfaces were taken with a Veeco Nanoscope Dimension 3100, working in tapping modeand using phosphorous doped n-type silicon tips \(Micromasch, San Jose, CA, USA).