Patent Application
20080274917
The invention described herein relates to surface activation of polymeric substrates. Particular embodiments provide methods for surface activation of polycarbonate substrates suitable for production of biochip platforms and detection of DNA and/or other biomolecules thereon.
Chip-based biosensors, such as DNA microarrays for example, have attracted increasing interest due to the many benefits of device miniaturization and parallel biomedical analysis. Such biosensors have typically been fabricated on glass, silicon or noble metal surfaces. Synthetic polymers may provide alternative substrates because of their low specific gravity, high elasticity and low cost. In the past, nylon membranes have been used to make DNA microarrays. Such nylon membranes suffer from the drawback that they exhibit lateral wicking characteristics, and attached DNA probes tend to spread from the points of immobilization. Other surface modification methods that involve comprehensive organic synthesis and fabrication steps, such as using graft polymer coating on silicon or gold surfaces, have been recently reported. Polycarbonate (PC) is an important thermoplastic because of its high optical clarity, tensile elongation and impact strength in comparison to many other materials. PC forms the base material for the manufacture of machine-readable optical discs (e.g. CDs, DVDs and the like), which are typically fabricated using inexpensive injection molding processes. In addition to being popular information storage media, optical discs have proven to be versatile tools/platforms for materials chemistry and biomedical research (see References 7-17). For example, Madou and co-workers have focused their efforts on the fabrication of microfluidic devices on circular plastic discs, which integrate microfluidic functions with CD technology, particularly the control of fluid transfer by disc spinning (see References 8 and 9) Several organizations, including Burstein Technologies, Gyros AB, and Tecan, have been working on the commercialization of similar devices; the Swedish firm Amic AB provides special optical disc fabrication services (e.g. preparation of high-precision masks and development of replication techniques).
Surface activation refers generally to procedures that convert relatively chemically inert surfaces of solid materials to be relatively reactive toward biomolecules of interest. In the past, the activation of polymer surfaces has relied primarily on prolonged ultraviolet (UV) irradiation. Liu et al. irradiated PC with a UV lamp (4 W, 220 nm, 5 hours exposure) to improve the aqueous fluid transport in microchip capillary electrophoresis devices and to facilitate the DNA probe attachment to different plastic substrates (polystyrene (PS), PC, poly(methylmethacrylate) (PMMA) and polypropylene) in microfluidic channel arrays (see References 28 and 29). Welle and Gottwald studied the effects of UV irradiation (low pressure 15-W UV lamp, at 185 and 254 nm) of PS, PMMA and PC on cell adhesion in vitro (see Reference 30). McCarley and co-workers prepared polymer-based microanalytical devices by “mild” UV activation (15 mW/cm2 at 254 nm) of PMMA and PC, and described their surface characteristics in detail (see References 31, 32 and 33). Recently, Kimura reported a simple, direct immobilization method: UV-induced attachment of poly(dT)-modified DNA strands to PC, PMMA, and polyethylene terephthalate (PET)—see Reference 34. These studies have demonstrated that UV light successfully converts PMMA to a bioreactive substrate with a high surface density of functional groups, leading to satisfactory results for DNA immobilization/hybridization. In contrast, only limited success has been achieved for PC, primarily. Without wishing to be bound by any particular theory, the prior art suggests that the limited success of activating PC substrates may be because of the low surface density of reactive groups, auto-fluorescence, and/or strong non-specific adsorption (see References 29, 31 and 32).
In view of the above, there is a general desire to provide methods for surface activation of PC substrates. Such methods may be used to convert PC substrates to polymeric platforms for the fabrication of chip-based biosensing devices, such as DNA microarrays, for example.
One aspect of the invention provides methods for surface activation of a surface comprising polymer chains. The methods comprise providing an ozone enriched environment in a vicinity of the surface and irradiating the surface with UV radiation.
Another aspect of the invention provides methods for conducting a biological assay on a surface. The method comprises: activating the surface by providing an ozone enriched environment in a vicinity of the surface and irradiating the surface with UV radiation; after activating the surface, allowing a first substance to react with molecules on the surface, thereby immobilizing the first substance on the surface; after immobilizing the first substance on the surface, allowing a second substance to come into contact with surface; and ascertaining whether there is a chemical reaction between the first and second substances.
Another aspect of the invention provides methods for the activation of PC substrates (such as the plastic bases of optical discs, for example). PC substrates can be readily converted to a polymeric platform for the fabrication of chip-based biosensing devices (DNA microarrays, for example) by treatment of the PC substrate using a combination of UV radiation and ozone reaction. The surface activation methods may be relatively rapid (less than 10 min) and efficient (yielding a high surface density of —COOH) when compared to prior art surface activation techniques used on PC. In comparison to prior art surface activation techniques, surface activation using the combination of UV radiation and ozone reaction is relatively non-destructive (i.e. the surface morphology of the PC substrate is not substantially altered).
Another aspect of the invention provides methods for fabricating bioanalytical devices by direct immobilization of DNA probes via photo-patterning/coupling reactions and by creating hybridization microarrays with microfluidic channel plates. The fabrication procedure (activation, patterning, and coupling) is simple and effective, and the resultant hybridization is highly sensitive and selective. Both passive and flow-through immobilization/hybridization experiments with various DNA probe-target complements have successfully detected single base-pair mismatches and reduced non-specific adsorption.
Methods described herein extend beyond the exemplary applications presented in this document. For example, the methods described herein are potentially useful for the development of disposable plastic biochips and the fabrication of biomedical devices that are readable with conventional CD drives.
Other features and aspects of specific embodiments of the invention are described below.
In drawings, which illustrate features of non-limiting embodiments of the invention:
Throughout the following description, specific details are set forth in order to provide a more thorough understanding of the invention. However, the invention may be practiced without these particulars. In other instances, well known elements have not been shown or described in detail to avoid unnecessarily obscuring the invention. Accordingly, the specification and drawings are to be regarded in an illustrative, rather than a restrictive, sense.
Particular embodiments of the invention provide methods for activating polycarbonate (PC) surfaces which involve irradiating the PC surface with UV radiation in the presence of ozone (O3) so as to cause a reaction between the irradiated PC surface and the ozone. The ozone may be present at the reaction site in a concentration greater than a concentration threshold. The ozone concentration threshold is greater than the ozone concentration at the surface of the earth (i.e. on the order of ˜0.3 ppm). In some embodiments, the ozone concentration is greater than 10 ppm. In some embodiments, the ozone concentration is greater than 20 ppm. In some embodiments, the ozone concentration may be higher.
The PC substrate may be irradiated at a UV radiation level of intensity level sufficiently low to avoid damaging the irradiated surface. In some embodiments, the radiation intensity is less than about 50 mW/cm2. In other embodiments, the radiation intensity is less than about 20 mW/cm2. In some embodiments, the UV radiation intensity may be lower.
Aspects of the invention also provide methods for producing biochip platforms on PC surfaces activated by UV radiation in the presence of ozone and for detection of DNA and/or other biomolecules thereon. Particular embodiments of the invention provide methods for photopatterning PC surfaces and for passive and/or microfluidic DNA immobilization/hybridization thereon. PC surfaces may be provided by standard optical discs (e.g. CDs, DVDs or the like). In some embodiments, DNA immobilized and/or hybridized on the PC surfaces of such optical discs according to the invention may be read in standard optical disc drives. Surface Activation and Characterization. In accordance with one particular embodiment of the invention, the PC base of an optical disc (or other suitable PC substrate) is activated by: (i) irradiation with UV radiation; and (ii) reaction with ozone. Preferably, ozone is present at the reaction site (i.e. at or near the PC surface) in a concentration greater than a concentration threshold. The ozone concentration threshold is greater than the ozone concentration at the surface of the earth (i.e. on the order of ˜0.3 ppm). In some embodiments, the ozone concentration is greater than 10 ppm. In some embodiments, the ozone concentration is greater than 20 ppm. In some embodiments, the ozone concentration may be higher.
The PC substrate may be irradiated at a UV radiation level of intensity level sufficiently low to avoid damaging the irradiated surface. In some embodiments, the radiation intensity is less than about 50 mW/cm2. In other embodiments, the radiation intensity is less than about 20 mW/cm2. In some embodiments, the UV radiation intensity may be lower.
Ozone may be introduced to the reaction site in any suitable manner. In some embodiments, UV radiation is used both to create ozone from O2 which may be present at the reaction site (e.g. by photolysis of molecular oxygen (O2)) and to irradiate the PC surface. Such UV radiation may be provided at different wavelengths (i.e. one wavelength that tends to promote the formation of ozone from molecular oxygen (O2) and a second wavelength that tends to promote the activation reaction at the PC surface. In other embodiments, ozone may be introduced to the reaction site by a secondary ozone source. By way of non-limiting example, a suitable secondary ozone source is the OZO-2VTT ozone generator sold by Ozomax, Inc. of Shefford, Quebec, Canada) and to provide the independently generated ozone in the presence of the PC surface.
In accordance with reaction methodology 101, PC (represented using the notation RH to indicate a hydrogen site on the PC molecule and using the reference numeral 102) is irradiated with UV 104 in the presence of ozone 106. At wavelengths between 254 and 300 nm, PC is known to undergo a photo-Fries reaction that results in the formation of phenyl salicylates and hydroxybenzophenones. The presence of ozone may induce the formation an O2-contact charge transfer complex (adduct 108), which is the initial step in the photo-oxidation of aliphatic and aromatic alkenes. Together, UV irradiation 104 and ozone 106 are thought to cause the formation of the adduct 108, where the notation “---” is used to refer to bonding between the carbon and oxygen atoms in these intermediate states. Adduct 108 is then thought to reassemble itself to forms a carboxylic group via a series of hydroperoxide intermediates. In the particular reaction mechanism shown in
In one particular exemplary embodiment of the invention for which experimental data has been obtained, the UV radiation was provided at about 1.5 mW/cm2 at 185 nm and at about 13.2 mW/cm2 at 254 nm (for a total combined UV radiation at about 15 mW/cm2) and the steady state ozone concentration was determined to be in a nominal range of approximately 25-75 ppm. It should be understood that these radiation wavelengths, radiation wavelengths and ozone concentrations are particular to the experimental apparatus and that other wavelengths, intensity levels and ozone concentrations could be used. In this exemplary embodiment, the low wavelength UV radiation (185 nm) is thought to cause the photolysis of molecular oxygen (O2) to form ozone at or near the PC surface and the higher wavelength UV radiation (254 nm) is thought to promote the surface activation reaction (e.g. reaction 101) at the PC surface. In some embodiments, the wavelength of UV radiation used to promote the generation of ozone from molecular oxygen (O2) is less than 240 nm and the wavelength of UV radiation used to promote the reaction mechanism shown in
UV radiation at the intensity level of about 15 mW/cm2 is generally considered to be “low power” or “mild” in comparison to prior art surface activation techniques as radiation at this intensity level does not cause significant damage to the irradiated PC surface. It will be appreciated by those skilled in the art that increasing the UV radiation intensity may increase the reaction rate at the PC surface (i.e. decreasing the time required to achieve a desired surface activation level), but if the UV radiation intensity is too high, there may be damage to the PC surface. This represents an engineering trade-off which may be tailored to suit particular applications.
A surface with a relatively low water contact angle may be said to be relatively hydrophilic, whereas a surface with a relatively high water contact angle may be said to be relatively hydrophobic. Reported reaction durations for using UV radiation alone (i.e. without ozone enrichment) to obtain hydrophilic PC surfaces vary significantly (from 60 min to more than 10 hrs), depending, for example, on the wavelength, power, and separation distance of the UV source.
In addition to the observed rate acceleration,
One difficulty associated with the use of surfaces activated using prior art techniques for various applications is the so-called “aging effect”, wherein the hydrophobicity of the activated surface increases (i.e. the surface activation level decreases) during sample storage. Without wishing to be bound by theory, it is believed that reorganization of the polymer chains on surfaces activated using prior art techniques induces the hydrophilic groups to move into the bulk of the substrate.
As shown in
The inventors also performed a number of contact angle titrations to confirm the formation of reactive carboxylic acid (—COOH) groups rather than other polar functionalities (e.g. alcoholic —OH) that tend to reduce the surface hydrophobicity of the PC surface.
The
The separation of the two “plateaus” in the
The inventors examined the surfaces of the PC substrates (e.g. optical disc bases) using taping-mode atomic force microscopy (AFM) to determine whether the topography of the PC substrates was significantly altered by the combination UV/ozone treatment.
Comparing
The combination UV/ozone surface activation processes described herein are substantially faster (short reaction time), more effective (the activated surface is more hydrophilic and the hydrophilicity lasts for a longer period of time), and non-destructive when compared to previous UV-irradiation-only activation methods.
Photo-Patterning and Passive DNA Immobilization/Hybridization.
Upon generation of reactive carboxylic acid groups in accordance with the activation processes described herein, the optical disc surface is converted into an effective platform for the construction of biochips. The inventors have demonstrated this application by the immobilization of DNA probe strands on the activated PC surface and subsequent hybridization with target samples using incubation techniques (e.g. by immersion of the activated PC substrates in bulk samples of modified DNA strands for coupling and hybridization). Other biological macromolecules (such as, by way of non-limiting example, protein, antibodies/antigens and carbohydrates) could be immobilized in a similar manner for various bioassay applications.
In one particular implementation for which experimental data was obtained, a PC base was first treated with a combination UV/ozone activation wherein the UV radiation was applied for 10 minutes at wavelength(s) of 254 nm and 185 nm at a power of about 15 mW/cm2 and ozone was present at a nominal level of 55 ppm. The UV radiation was applied to the PC surface through transmittance electron microscopy (TEM) grids, which were used as photomasks over the PC surface to achieve surface activation and micro-patterning of the PC surface in a single step. DNA probe strands were then attached to the activated and micro-patterned PC surface. To attach specific DNA probe strands (listed in Table 1) modified at their 5′-ends with amino groups via C6 linkers, amide linkages were formed via a 1-ethyl-3-(3′-dimethylaminopropyl)-carbodiimide (EDC)/N-hydroxysuccinimide (NHS) coupling reaction, as described in Reference 46 which is hereby incorporated herein by reference.
AAA ACT TAA A-3′
In contrast to
As an experimental control, single-base mismatched and non-complementary amino-terminated DNA probes were tested using the same procedure.
Microfluidic DNA Microarrays on PC. The inventors used microfluidic methods to create DNA microarrays on PC surfaces activated using the combination UV/ozone surface activation technique to demonstrate the application of the UV/ozone surface activation technique for the activation of plastic materials in biochip fabrication. Microfluidic methods for creating DNA microarrays on surfaces are described in References 26, 27, 32 and 48, which are hereby incorporated herein by reference. In comparison with traditional DNA microarray fabrication procedures (either by on-chip photolithographic synthesis of DNA probes or by robotic spotting of pre-synthesized oligonucleotides, for example), microfluidic methods for creating DNA microarrays are simple and do not require expensive facilities or instrumentation.
In order to examine the efficiency of flow-through immobilization of DNA probes on PC substrates, the inventors used different concentrations of a marker strand modified with an amino group at the 5′-end for surface coupling and with a fluorescent tag (Cy5) at the 3′-end for imaging (see Table 1). The results of this examination are shown in
While not wishing to be bound by any particular theory, the rate of surface coupling (i.e. of DNA to the activated PC surface) may be limited by the diffusion of DNA molecules to the surface; this appears to be the primary reason for the more efficient immobilization in the flow-through setting. A larger amount of DNA molecules can be transported to the PC surface by convection (flow) than by passive incubation. This interpretation is supported by the depletion effect, i.e., the apparent “fading” of the probe line at low marker concentrations (
The efficient immobilization of single-stranded DNA (ssDNA) probes led the inventors to test the hybridization with specific DNA targets using the microfluidic technique, assuming that non-specific adsorption of DNA strands can be minimized.
As shown in
Using immobilization and hybridization experiments, the inventors have determined that the overall uniformity of hybridization spots can be easily repeated from one chip to another. The thermal and chemical stability of immobilized DNA probes created on the PC substrates have been examined by varying the temperature (up to 90° C.) and salt concentrations (up to 0.1 M). Such high temperatures and high salt concentrations did not result in significant changes (i.e., about 2% variation after each cycle) in the hybridization capabilities.
In addition to the choice of buffer solution, the inventors also systematically investigated the effect of target concentration on the hybridization results (e.g. the lowest concentration that produces detectable signal that is measurably different than the background noise (for example, three times greater than the background noise is a current industry standard))−also referred to as the detection limit).
Based on the effective probe immobilization and on-surface target hybridization, the inventors performed a DNA identification assay to evaluate the hybridization of the same DNA target strand with three different probe strands that were immobilized with the first PDMS channel plate. The results of this experiment are shown in
Before hybridization, fluorescence was not detected on the chip (data not shown). As shown in
Although the experimental conditions can be further optimized to achieve higher sensitivity, the above-described results demonstrate the feasibility of creating DNA hybridization microarrays on PC substrates after surface activation by a combination of UV irradiation and ozone reaction and illustrate how the inventors' surface activation methods can be applied to the preparation of bioreactive substrates for the fabrication of microanalytical devices.
The following section describes particular non-limiting techniques, supplies and equipment used by the inventors to carry out the specific experiments described above.
Surface Activation and Characterization. In the experiments described above, polycarbonate (PC) bases of optical discs were provided by Millennium Compact Disc Industries Inc. (Vancouver, BC, Canada), or prepared from regular optical discs by: removing the reflective layer via scoring and vigorous rinsing with deionized water; removing the dye layer with a rapid methanol rinse, 10-min ultrasonication in 1:4 (v/v) methanol/water; and providing a final rinse with deionized water (see Reference 20 which is hereby incorporated herein by reference). The DNA oligomers (sequences listed in Table 1) were of reverse-phase cartridge purification (RP1) grade and obtained from Sigma-Genosys (Oakville, ON, Canada).
The PC surfaces were activated using the combination of UV radiation and ozone reaction as discussed above using a UV irradiating system (Model PSD-UV) from Novascan Technologies, Inc. (Ames, Iowa, USA). This apparatus uses a low-pressure mercury lamp and generates UV emission at two wavelengths (185 nm (1.5 mW/cm2) and 254 nm (13.2 mW/cm2) with a total power of about 15 mW/cm2.
Water contact angles on activated PC surfaces were measured with an AST Optima system at ambient conditions (22-26° C., 43±3% relative humidity) using a horizontal light beam to illuminate the liquid droplet. The contact angles described above are the values of sessile liquid drops of either pure water or aqueous buffer solution. The surface topographies of the pristine and the UV/ozone-treated PC surfaces were examined with an MFP-3D-SA Atomic Force Microscope from Asylum Research, Inc. (Santa Barbara, Calif., USA) in tapping mode. Root-mean-square (RMS) roughness factors were calculated using the IGOR Pro 4 software provided by the manufacturer.
Photo-patterning and Passive DNA Immobilization/Hybridization. After the activation process using combined UV radiation and ozone reaction, 10 μl of a 10 μM solution of DNA probe strands in 0.1 M MES (2-(N-morpholino) ethanesulfonic acid) buffer at pH 6.5 (also containing 5 mM EDC (1-ethyl-3-(3′-dimethylaminopropyl)carbodiimide) and 0.33 mM NHS (N-hydroxysuccinimide)) were spread onto the patterned PC surface (a TEM grid was placed on top during UV/ozone treatment). The sample was incubated for 2 hours under ambient conditions. For the hybridization test, a 10 μM solution (10 μl) of fluorescein-labeled DNA target strands (0.1 M MgCl2 and 1 M NaCl in 10 mM Tris-HCl buffer) was spread onto the surface. After immobilization, DNA oligomers immobilized on the PC base of the optical disc were imaged with a Zeiss LSM 410 confocal microscope (Oberkochen, Germany) equipped with krypton/argon laser sources. Creation of Microfluidic DNA Microarrays on PC. The PDMS microchannel plates were prepared following the standard procedure reported in the literature (see Reference 52 which is hereby incorporated herein by reference). The first channel plate (with 8 to 12 channels) was sealed with the activated PC for DNA probe immobilization. Probe DNA samples (typically 0.5 μl were injected into the channel reservoirs on one side and passed through the channels by suction from the other ends of the channels. The solution was allowed to stay in the channel for 5-10 hours at room temperature for DNA probe immobilization. To wash, Tris buffer was passed through the channels at least three times. Then the PDMS plate was peeled off, and the substrate was treated with glycogen solution to reduce (potential) nonspecific adsorption. Afterward, the second PDMS chip was placed on top of the substrate but in a substantially perpendicular orientation. Hybridization was done by using Cy5-labeled DNA strands (0.1-2 μM); this step took place in a humid box at 20-40° C. for 30 min. After hybridization, the PDMS plate was peeled off, and the PC surface was washed with buffers and dried with nitrogen gas. The PC surface was then scanned using a Typhoon 9410 confocal laser-fluorescence scanner available from Amersham Biosystems (now GE Healthcare) at a resolution of 25 μm. Radioisotope labeling measurements. 50 pmol 5′-modified ssDNA and 100 pmol DNA template with a two-nucleotide-5′ overhang (3′-TG-5′) were hybridized in 5 μl buffer (50 mM Tris at pH 7.2, 10 mM MgCl2, 0.1 mM DTT, 1 mg/ml BSA) by heating at 90° C. for 2 min, followed by immediate cooling with ice. The labeling reaction was started by adding 5 μl of the above buffer, containing 1 nmol dATP, 6.67 pmol [α-32P]dATP (3000 Ci/mmol, 10 mCi/ml) and 2 U Klenow fragment of DNA polymerase I (Roch, Mannheim, Germany). After 2 hours, the labeled oligonucleotides were purified by precipitation with ethanol followed by 20% denaturing polyacrylamide gel electrophoresis (PAGE). The procedure for the modification and washing of the PC surface with 32P-radiolabeled DNA was the same as other DNA strands (without radiolabeling). The DNA surface density was calculated by comparing the radioactivity of DNA immobilized on a certain area of the PC surface with that of a known amount of DNA. For this purpose, two control samples having known amount of 32P-radiolabeled DNA were dropped on two reference PC surfaces and allowed to air-dry without any washing. The radioactivity was read by phosphor imaging using the Typhoon 9410 scanner.
Thermal/chemical stability tests. PC substrates with immobilized DNA probes were treated under PCR (Polymerase chain reaction)-like conditions (alternate immersions of the chip into three buffers at different temperatures) for up to 10 cycles. Each cycle consisted of a “denaturing step” at 90° C. for 30 s, an “annealing step” at 50° C. for 30 S and an “extension step” at 72° C. for 30 s. After each cycle of the treatment, the slides were washed and used for hybridization experiments as described above.
The following section provides additional, non-limiting information relevant to particular experiments conducted by the inventors.
Reagents and materials. 1-Ethyl-3-(3′-dimethylaminopropyl)carbodiimide (EDC, water-soluble carbodiimide), N-hydroxysuccinimide (NHS) and glycogen (Type III, from rabbit liver) were purchased from Sigma-Aldrich (St. Louis, Mo.), 2-(N-morpholino)ethanesulfonic acid (MES) from Fluka (Buchs, CH), sodium chloride, tris(hydroxymethyl)aminomethane, sodium citrate, sodium dodecylsulfonate (SDS), and magnesium chloride from Calcdon Laboratories Ltd (Georgetown, ON). All chemicals used without further purification unless otherwise stated. All solutions were prepared with deionized water (>18.3 MΩ·cm) from a Barnstead EasyPure UV/UF compact water system (Dubuque, Iowa). The 22-mer synthetic oligonucleotides used as probe/target strands in the experiments were of reverse-phase cartridge purification (RP1) grade and obtained from Sigma-Genosys (Oakville, ON).
Polycarbonate (PC) bases of compact discs (CDs) were provided by Millennium Compact Disc Industries Inc. (Vancouver, BC). They can also be prepared from regular CDs (or CD-Rs or the like) by removing the reflective layer via scoring and vigorous rinsing with deionized water, removal of the dye layer with a rapid methanol rinse, 10 min ultrasonication in a 1:4 (v/v) methanol/water solution and a final rinse with deionized water (see Reference 20). Transmittance electron microscopy (TEM) gold grids (G1000HSG, Pelco International) were used as masks for patterning the PC surfaces. They were made of 6-μm-diameter wires with a center-to-center spacing of 25 μm.
Activation and patterning of the PC substrates by UV/ozone treatment. Without wishing to be bound by any particular theory, it is thought that the photochemistry of polycarbonate exposed to UV light involves two photo-Fries reactions, a photo-induced oxidation of the side-chain and a benzene ring oxidation. The reaction pathway followed is thought to depend primarily on the excitation light source and the oxygen concentration. The main photochemical process occurring under irradiation at 254 nm in the presence of oxygen may be the succession of two photo-Fries rearrangements leading to the formation of phenyl salicylate and dihydroxybenzophenone units. Competitively, some radicals may react with oxygen to form hydroperoxides. Eventually the photo-oxidation leads to the formation of carboxylic acid groups. Particular theories as to the photochemistry of PC exposed to UV light are described in References 35, 42, 54 and 55.
The UV/ozone treatment of the PC surface was carried out with a UV/ozone system (Model PSD-UV, Novascan Technologies, Inc.). This apparatus uses a low-pressure mercury lamp, generating ultraviolet emission at both 185 nm and 254 nm with a total power measured to be about 15 mW/cm2; the distance between the UV source and the PC sheet was 2.5 cm. In the presence of ambient oxygen, the two-step photochemical process initiated by the photolysis of molecular oxygen (O2) at 185 nm produces a nominal steady-state concentration of highly reactive ozone which then decomposes by absorption of UV light at 254 nm.
Contact angle measurements of UV/ozone-treated PC surface. Contact angle measurement is one convenient technique for characterization of solid/liquid interfaces. Water contact angles on activated PC surfaces were measured with an AST Optima system at ambient conditions (22-26° C., 43±3% relative humidity) using a horizontal light beam to illuminate the liquid droplet. The contact angles described above are equilibrated values of sessile liquid drops of either pure water or buffer solution.
The untreated PC substrate is hydrophobic with a water contact angle of 88±2°. During UV/ozone treatment, the surface became more and more hydrophilic (see
For the contact angle titration (see
Determination of the surface density of carboxylic acid groups on activated PC. To determine the surface density of carboxylic acid groups (COOH) groups resulting from UV/ozone treatment, a cationic dye, crystal violet, was used. This method makes use of the electrostatic interactions between crystal violet molecules and carboxylate groups. First, the UV/ozone-irradiated substrates were covered with a crystal violet solution (1 mM) for 5 min. After rinsing with water, the samples were incubated first with ethanol (80% v/v) and second with 0.10 M HCl (in 20% ethanol) until the dye could no longer be observed on the sample surface. Then the solutions from the two incubations were combined and absorbance readings were taken with a UV/Vis spectrometer. The concentration of crystal violet released from the surface was calculated from Beer's law (A=εcl) and used to determine the surface density of COOH groups. The reported value of 4.8±0.2×10−10 mol/cm2 represents an average over three samples.
XPS confirmation of the carboxylic acid groups generated on PC upon UV/ozone treatment. The generation of reactive carboxylic acid groups was further confirmed by x-ray photoelectron spectroscopic (XPS) studies of three types of PC samples: original, UV/ozone-treated, and UV/ozone-irradiated through a TEM grid. The characteristic C 1s and O 1s signals are shown in
Comparison of the surface activation efficiency by different UV irradiation methods. As shown in Table 2, the surface density of active groups (and the surface wettability) on polymeric materials upon UV treatment depends on various irradiation conditions (such as wavelength, powder, and duration). Compared with other UV irradiation methods, the UV/ozone methods described herein show higher surface activation efficiency (i.e. shorter reaction time and higher —COOH surface density), especially for PC substrates.
Photo-patterning of PC and passive DNA immobilization/hybridization.
For the particular experiments described herein, after hybridization the PC chips were imaged on a Zeiss LSM 410 (Oberkochen, Germany) confocal microscope equipped with a ×25 (NA 0.8) multi-immersion objective. An argon/krypton mixed gas laser with excitation lines at 488, 568, and 647 nm was used to induce fluorescence. Excitation of the green fluorophore was achieved at 488 nm (the effective excitation range of 488-495 nm for fluorescein closely matches the photo-emission of an argon laser), and the resulting fluorescence was observed by using a 515-540 nm band pass filter.
Creation of DNA microarrays with microfluidic channel plates. A small PDMS plate with 8 to 12 microchannels (300 μm wide and 25 μm deep) was laid on top of an activated PC substrate. The probe solution (0.5 μL) containing 5′-amine-modified DNA molecules (10-50 μM in phosphate buffer, 0.10 M, pH 7.0) was injected into the reservoir on one terminal of a microchannel and passed through the channel by suction from the other end. After 10 hours incubation in a humid box at room temperature, the channel was washed with the phosphate buffer.
The PDMS plate was then peeled off from the PC substrate. The surface was “blocked” with glycogen and washed again with the phosphate buffer. Another PDMS plate was then laid on top of the PC surface, but in a substantially perpendicular orientation with respect the first plate. Hybridization with Cy5-labeled DNA samples (1-2 μM) in pH 7.4 buffer (10 mM Tris, 500 mM NaCl, 50 mM MgCl2) or in ×1 SSC pH 7.0 buffer (15 mM Na2C2O4, 150 mM NaCl, 0.15% SDS) was carried out at 20-40° C. for 30-60 min.
After hybridization, the microchip was washed sequentially with three buffers (pH 7.4): Tris (10 mM)+NaCl (50 mM), Tris (10 mM)+NaCl (10 mM), and Tris (10 mM) only. If a SSC buffer was used in the hybridization experiment, it was also used to wash the microchip twice. Afterwards, the PC chip was rinsed with water and dried with nitrogen gas. A confocal laser-fluorescent scanner (Typhoon 9410, Amersham Biosystems) at a resolution of 25 μm was used to examine the efficiency of marker strand immobilization and of the hybridization.
As will be apparent to those skilled in the art in the light of the foregoing disclosure, many alterations and modifications are possible in the practice of this invention without departing from the spirit or scope thereof. For example:
This application claims the benefit of the priority of U.S. Application No. 60/882,392 filed Dec. 28, 2006 and U.S. Application No. 60/878,770 filed Jan. 5, 2007, both of which are hereby incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| 60882392 | Dec 2006 | US | |
| 60878770 | Jan 2007 | US |
