Feasibility of using carboxylic-rich polymeric surfaces for the covalent binding of oligonucleotides for microPCR applications

Please download to get full document.

View again

of 9
6 views
PDF
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Document Description
Feasibility of using carboxylic-rich polymeric surfaces for the covalent binding of oligonucleotides for microPCR applications
Document Share
Document Tags
Document Transcript
  I NSTITUTE OF  P HYSICS  P UBLISHING  S MART  M ATERIALS AND  S TRUCTURES Smart Mater. Struct.  11  (2002) 783–791 PII: S0964-1726(02)52472-1 Feasibility of using carboxylic-richpolymeric surfaces for the covalentbinding of oligonucleotides for microPCRapplications Elena P Ivanova 1 , Michal Papiernik 1 , Anna Oliveira 1 ,Igor Sbarski 1 , Thomas Smekal 2 , Piotr Grodzinski 2 andDan V Nicolau 1 1 Swinburne University of Technology, PO Box 218, Hawthorn, Vic 3122, Australia 2 Microfluidics Laboratory, Motorola Physical Sciences Research Labs, Phoenix,AZ83/ML34, USAE-mail: eivanova@swin.edu.auand dnicolau@swin.edu.au Received 5 June 2002, in final form 3 July 2002Published 20 September 2002Online at stacks.iop.org/SMS/11/783 Abstract The chemical binding of oligonucleotide/DNA on polystyrene-relatedpolymeric surfaces has been investigated using contact angle measurements,x-ray photoelectron spectroscopy and gravimetric analysis. The results of the covalent attachment of the phosphorylated oligonucleotides using thehetero-bifunctional cross linker1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride topolystyrene-co-maleic anhydride (PSMAA) and polystyrene-co-maleic acid(PSMA) are described. The immobilization efficiency of covalently coupled26-mer oligonucleotides to polymeric surfaces was estimated as 0.3 × 10 10 and 0.1 × 10 9 molecules mm − 2 for PSMA and PSMAA, respectively. Theresults suggest that, although the covalent binding on PSMAA  per se  is notcapable of the high density of DNA required by micro-PCR applications, themethod has the potential to be used as a cheap alternative for other low-cost,less DNA-sensitive applications such as disposable biosensors. 1. Introduction The emergence of various DNA-based micro-applications,such as high-density miniaturized oligonucleotide arrays[1–4], disposable biosensors [5–7] and lab-on-the-chip devices [8–10], has translated into intensive efforts focusedon DNA-modified surfaces in recent years. The developmentof the robust solid supports useful for stable, spatiallyresolved and efficient DNA immobilization is critical forDNA chip technology. This emerging and varied technologyrequires a multitude of substrate materials, with an increasingpreference for polymeric materials, both because of itsdiverse applications and its immature stage of development.Therefore, many of the technical difficulties are relatedto the lack of a ‘magic’ surface chemistry that wouldapply to a large number of possible solid supports, whichwould assure a high, reproducible and controllable surfaceconcentrationofoligonucleotide/DNA.Duetotheirversatility,microlithography-basedmaterialscouldbe—inprinciple—thebase of a good strategy, as their thermo-mechanical propertiesand surface chemistry can be controllably altered by exposureto light or another form of radiation [11–13]. However,microlithography is a mature technology with extremelyspecialized equipment and materials, which come with a highproduction cost. Nevertheless, as many microlithographicmaterialsstartedassimpleformulations ofcommon polymers,and the surface-based chemistry is increasingly central tomicrolithography, this technology can be used at least as atestbed for the search for the appropriate strategy towards amaterial-specific oligonucleotide/DNA functionalization. 0964-1726/02/050783+09$30.00 © 2002 IOP Publishing Ltd Printed in the UK  783  E P Ivanova  et al Figure 1.  Envisaged chemistry on the PSMAA surface afteracidic/basictreatment. DNA fragments (oligonucleotides) are attached onsurfaces mainly through two very different methods, i.e.eitheradsorption or chemical binding. In the first mechanism, theattachment of the DNA fragments on surfaces is governedby purely physical phenomena that are not highly specificand are often reversible. It follows that simple adsorption isunlikely to fulfil the increasingly strict requirements for thefuture DNA-based microdevices. In the second mechanism,which is mainly a chemistry-controlled process, the DNAfragments are covalently bound to surfaces. There are manywell known chemical pathways for the covalent bonding of DNA on polymer surfaces [14–16]. Of the possible covalent binding sites, the OH-, NH 2 -, COOH-(or related groups) andSH-functionalizedsurfaceshavecommonlybeenused[17,18]. However, the availability of the binding sitesin common, low-cost polymers is not evenly spread, with oxygen-containinggroups (e.g. COOH) being far more common than other (NH 2 or SH) groups. Moreover, for micro-PCR applications, whichis the main focus of the present study, the pool of feasiblepolymerswithregardtothermo-mechanicalpropertiesandcostis even further restricted.To this end, this study explores the possibilities leadingto usage of low-cost polystyrene-derived polymers that wouldhave appropriate thermo-mechanical characteristics and satis-factory covalent binding chemistries for oligonucleotide/DNAattachment. 2. Materials and methods 2.1. Screening polymers for microPCR applications The polymers in a polymer database (from SciPolymer 3.0,SciVision) have been screened for the possible use for amicroPCRdeviceinaccordancewiththeparametersdetailedinthe results and discussion section. Of the 660 homo-polymerspresent in the database, 42 have been selected as chemicallypossible candidates (table 1). 2.2. Polymeric surface preparation and functionalization Poly(styrene- co -maleic anhydride)—PSMAA—and poly(styrene- co -maleic acid)—PSMA (MW  ∼  225000)—werepurchased from Sigma-Aldrich. CovaLink  TM , NucleoLink  TM and Top Yield TM strips, commonly used for oligonucleotidecovalent binding, were purchased from Nalge NuncInternational and used as benchmark polymeric surfaces. Allof the samples were used without any further treatment.PSMAA was dissolved in toluene (99.7%) and PSMA wasdissolved intetrahydrofurane (TTF) (99.9%) to reach polymersolutions of 5 mg ml − 1 . First, 30 mm  ×  50 mm glasssubstrates were primed with hexamethyldisilazane (HMDS)via spin coating at 1000 rpm. Subsequently, the polymerswere spincoated on glass substrates from polymer solutions at3000rpmwitharampaccelerationof1000rpmmin − 1 . Finally,the samples were baked for 60 min at 95 ◦ C. To achieve thehydrolysis of the anhydride groups to acid groups, PSMAAslides were treated after baking using either 1 M HCl or 1 MNaOHsolutionsfor30minandthenwashedwithMilliQwater.The envisaged chemical reaction is presented in figure 1. 2.3. Measurement of the surface hydrophobicity The hydrophilicity/hydrophobicity of the spin-coated polymersurfaces was estimated using multiple measurements of thecontact angles for PSMAA, PSMA and HCl- and NaOH-treated PSMAA surfaces according to the static solvent–distilled water method on an NRL goniometer with an errorof  ± 2 ◦ as described elsewhere [12, 24]. The temperature was22 ◦ C, and the relative humidity was 60%. 2.4. X-ray photoelectron spectroscopy Chemical characterization of the surface modification chem-istry was performed using x-ray photoelectron spectroscopy(XPS) measurements. XPS spectra were obtained using anAl K α  sour c e (1486.9 eV photon energy) and a 16-channeldetector. 2.5. Thermogravimetric analysis Thephysicalcharacteristicsofthepolymers weremeasuredbydifferential scanning calorimetric thermogravimetric analysis(DSC-TGA)ina controlled atmosphere usingMettlerTA3000thermalanalysissystem. Boththeevolution ofweightandheatflux versus temperature were recorded. 2.6. DNA attachment to polymeric surfaces A 26-base-pair universal oligonucleotide primer 5 ′ GTG GATCAC CTG AGG TCA GGA GTT TC3 ′ correspondingto the ‘ alu  gene’ was used for covalent binding tofunctionalized polymeric surfaces. The  alu  gene primerbinds in both the forward and reverse directions, making itparticularly convenient for binding studies [25, 26]. Theprimer was phosphorylated at the 5 ′ -terminus and labelledwith the fluorescein isothiocyanate (FITC) as purchasedfrom GeneWorks at the 3 ′ -terminus. A multifunctionalcross linker, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC), was chosen because it is reactivetowards both NH 2  and COOH groups. Because of thesimilarity in chemical structure and character, EDC will alsoreact with DNA’s phosphate groups (PO − 4  ) . The chemistry of EDC/DNA attachment is presented elsewhere [23]. For DNAimmobilization on the polymeric surfaces, approximately 5 µ lof ice-cold 0.1 M 1-methylimidazole (1-MIA) was placeddirectly on the slide followed by the addition of 7.5  µ l of DNA solution. 25  µ l of freshly prepared EDC (0.2 M EDC in10mM1-MIA)wasfinallyaddedtothesuspensionontheslidesurface. Thesolutionwasthenincubatedforapproximately3hat 50 ◦ C toenable the DNA attachment ina sealedchamber (toavoid evaporation). The slides were then washed three times784  Covalent binding of oligonucleotideson carboxylic-rich polymers Table 1.  Physical parameters of some candidatesfor oligonucleotidefunctionalization. T  g  V  m  E  Coh  Sf TenChemical name (K) (cm 3 mol − 1 ) (J mol − 1 ) (dyn cm − 1 )Poly(1, 1-dimethylsilazane) 181 82.5 20457 21.6Poly(10-aminodecanoicacid) 284 169.3 79768 42.5Poly(11-aminoundecanoicacid) 277 185.4 84709 41.8Poly(12-aminododecanoicacid) 271 201.4 89650 41.3Poly(2, 2, 2 ′ -trimethylhexamethyleneterephthalamide) 448 263.2 142251 44.8Nylon 6, 12 292 306.4 149653 43.4Poly(hexamethyleneadipamide) 330 209.9 120006 47.6Poly(hexamethyleneazelamide) 308 258.2 134830 45Poly(hexamethyleneisophthalamide) 428 212.9 129722 49.5Poly(hexamethylenesebacamide) 302 274.2 139771 44.4Poly( m -phenyleneisophthalamide) 502 184 129223 54.5Poly( m -phenyleneterephthalamide) 536 184 129223 54.5Poly( m -xylyleneadipamide) 348 211.3 129389 52.9Poly( o -phenyleneisophthalamide) 474 184 129389 54.7Poly( o -phenyleneterephthalamide) 508 184 129389 54.7Poly(  p -phenyleneisophthalamide) 536 184 129223 54.5Poly(  p -phenyleneterephthalamide) 569 184 129223 54.5Poly(  p -xylylenesebacamide) 346 275.7 149154 48.2Poly[2, 2 ′ -( m -phenylene)-5,5 ′ -bibenzimidazole] 622 231.9 124100 55Poly[  N  -(2-phenylethyl)methacrylamide] 394 170.8 87795 47Polyacrylamide,PAA 366 56.2 44297 50.8Polyphenolphthalein1 576 421.7 244194 51.1Polyphenolphthalein4 625 501.8 290986 54.6Polyphenolphthalein5 632 504.6 308921 54.4Resin F 391 300 193834 48.2Torlon 587 275.3 165305 51.9Cellulose, Cel 440 112.2 112728 50.5Phenoxy resin 407 251.7 120877 40.7Poly(2-hydroxyethylmethacrylate),PHEMA 354 106.6 66502 42.8Poly(2-hydroxypropylmethacrylate),PHPMA 363 124.5 70018 40.3Poly( m -hydroxymethylstyrene), PHMS 390 117.9 69928 48.1Poly(oxy-2-hydroxytrimethylyne-oxy-1,4-phenyleneisobutylidene-1,4-phenylene) 411 268.6 126418 40.9Poly(oxy-2-hydroxytrimethylyne-oxy-1,4-phenyleneisopropylidene-1,4-phenylene) 407 251.7 120877 40.7Poly(  p -hydroxybenzoate) 433 91.1 46676 51.3Poly(  p -hydroxymethylstyrene) 390 117.9 69928 48.1Poly(vinyl alcohol) 333 36.7 35297 48.4Poly(maleic anhydride),PMAA 462 57.1 39831 74.7Poly(methacrylicacid), PMA 393 70.6 38748 44.2Poly(  p -methacryloxybenzoic acid), P  p MXBA 412 159.7 85424 50.5 withasolutionconsistingof0.4MNaOHand0.25%SDS(pre-heated to 50 ◦ C) and soaked for 5 min in the washing solution,before being washed again three times [23]. CovaLink  TM ,NucleoLink  TM and Top Yield TM strips were also used forcovalent attachment of the DNA as the controls according tomanufacturers’ instructions. 2.7. Analysis of the oligonucleotide attachment on polymericsurfaces The DNA functionalized polymeric films were visualizedand analysed using two different microscopic systems. Onewas the Nikon Microphot FX upright microscope with a UVlight source (Nikon mercury lamp, HBO-100 W/2; NikonC.SHG1 super-high-pressure mercury lamp power supply) at100 × objective. These images were captured on a Nikoncamera (FX-35WA). The second system was a Nikon invertedmicroscope (Nikon Eclipse TE-DH 100 W, 12 V) with anattached UV light source (Nikon TE-FM Epi-Fluorescence).Images were captured on a Nikon CCD camera. Thefluorescence intensities wereanalysed usingGel-ProAnalysersoftware, version 4.0, from Media Cybernetics. 3. Results and discussion 3.1. Selection of the polymers for microPCR applications The materials for microPCR applications should fulfil thefollowing logical requirements:(i) a melting temperature ( T  m ) well above the PCR processtemperature (95 ◦ C);(ii) optically transparent for optical detection;(iii) no, or very low (background), fluorescence;(iv) low coefficient of thermal expansion and(v) low cost of the material and its processing.The last requirement drastically limits the choice of polymers.For polymers, requirement (iv) actually imposes a quitehigh glass transition temperature ( T  g ), usually higher than120 ◦ C, which will automatically fulfil the first requirement.Fortunately, many polymers are transparent, in particularacrylicpolymers, whichtendtohavelower T  g . However,manypolymers are fluorescent, in particular aromatic polymers,which also present the tendency for higher  T  g ’s. Whateverthe material and the attachment method, the density of 785  E P Ivanova  et al Tg acceptable range 0100200300400500600100 200 300 400 500 600 700 Glass transition temperature, Tg (K)    M  o   l  a  r  v  o   l  u  m  e ,   V  m   (  c  m    3    /  m  o   l  e   ) VmNHVmOHVmCOOH   PpMXBA   PMAA   PMA   PHEMACellulosePHMS   PHPMAPAA Tg acceptable range Figure 2.  Dependence of the molar volume on glass transition temperature for the selected polymers (captions as in table 1). 1020304050607080100 200 300 400 500 600 700 Glass transition temperature, Tg (C)    S  u  r   f  a  c  e   t  e  n  s   i  o  n   (   d  y  n  e   /  c  m   ) STenNHSTenOHSTenCOOH Tg acceptable range PpMXBA   PMAA   PMA   Figure 3.  Dependence of the surface tension on glass transition temperature for the selected polymers (captions as in table 1). oligonucleotides on the surface should reach at least 3 × 10 7 molecules  µ m − 2 . These requirements could be in principlefulfilled by choosing an acrylic/aromatic copolymer.A separate discussion is needed regarding the surfacetension of the polymers. The surface hydrophobicity (high forlowsurfacetension)hasanimpactontheoligonucleotide/DNAsurfacedensitywithhigherhydrophobicities inducing ahighersurface density via the adsorption of the bases. However, thisparticular self-assemblyof the oligonucleotides/DNA withthebases facing down the surface [28] is detrimental to the PCRprocess, and itisalsoirrelevant ifa covalent binding method isused. However,ahigherhydrophilicitycouldbetheexpressionof the solubility or gel-like character of the polymer when incontact with aqueous solutions, which is totally detrimental to(micro)PCR devices. With these qualifications, a hydrophilicsurface should be preferred over a hydrophobic one.Regarding the procedures of the immobilization of theoligonucleotides/DNA on the surface, the easiest methodis to use physical adsorption, either based on hydrophobicattachment or on electrostatic interaction [27]. However, theratherharshandrepetitivecharacterofthe(micro)PCRprocesswould recommend a much stronger attachment, usuallyattained via covalent binding on the surface. The restrictionsregarding a cost-effective technical solution effectively meanchoosing the polymeric materials (or combinations of these)thatwouldhave covalentlybinding relevant groups inthebulk,andtherefore on thesurface. Asexplained inthe materials andmethods section, the logical choices would be to use polymerswithNH 2 , OHorCOOHgroups, presented intable1(42casesof homo-polymers).The first requirement is a high  T  g  and subsequently a high T  m . Also important, a lower coefficient of thermal expansionwill be expected for higher densities (lower molar volume).Thedistribution ofthesetwoparameters withinthe42selectedpolymers is presented in figure 2. In general, although theNH 2 -containingpolymersspanthewholerangeof  T  g ,theyalsopresent lower densities than both OH- and COOH-containingones. Moreover, the NH 2  present in the high- T  g  polymers isactually close to an aromatic ring, which would make it verydifficult to use for covalent binding. Only poly(acryl amide)786  Covalent binding of oligonucleotideson carboxylic-rich polymers Figure 4.  TGA analysis of PSMA polymer. Figure 5.  TGA analysis of PSMAA polymer. (PAA) seems to have the borderline right characteristics, butit was not used in this study due to its too high hydrophilicity.OH-containing polymers are also excluded, as they are eitherhydrogels (PHEMA and PHPMA) or highly inflammable anddifficult to process (cellulose). The only reasonable choice isCOOH-containing polymers, of which poly(  p -methacryloxybenzoic acid), P  p MXBA, is also excluded due to its low den-sity. The distribution of the surface tension versus  T  g  (fig-ure 3) shows that COOH-containing polymers are also amongthemosthydrophilic, although thesurface tensionofPMAAissomewhat exaggerated (estimated using group contributions).One last concern has been the processability of thepolymers. PMA and PMAA are not the best candidates formicro-moulding. To ensure a good processability, we havechosen to study the possibility of DNA functionalization of co-polymers with polystyrene, namely PSMAA and PSMA.We also have chosen three commercial polymersused for oligonucleotide surface functionalization, namelyCovaLink  TM , NucleoLink  TM and TopYield TM , as benchmark materials. CovaLink  TM is a relatively novel type of micro-well surface, described as having secondary amino groupspositioned at the end of spacer arms that are covalentlygrafted onto a polystyrene surface via a spacer. The linkergrafted onto CovaLink  TM has a density of approximately10 14 sites cm − 2 [23]. NucleoLink  TM is a polymer designedto use in PCR. The special feature of NucleoLink  TM is itsability to bind DNA by a covalent and heat-stable bond,with the binding described as being stronger than that of CovaLink  TM [23]. TopYield TM isa newlydeveloped polymerbased on a polycarbonate structure, with no propensity forcovalent binding. 3.2. Bulk and surface analyses of the selected polymers According to the thermogravimetric analysis, PSMA can beused without important changes up to 130 ◦ C, with the decar-boxylation of the surface starting at around 160 ◦ C (figure 4).On the other hand, the other polymer candidate, PSMAA,has a larger usage range, up to 200 ◦ C, when the oxidationis starting to occur (figure 5). These characteristics are simi-lar to those of CovaLink  TM , NucleoLink  TM and Top Yield TM strips (figures 6–8, respectively). CovaLink  TM is effectivelyunchanged up to 110 ◦ C, while NucleoLink  TM changes in anentirely similar manner with PSMA and PSMAA. Interest-ingly, after 140 ◦ C Nucleolink  TM starts to acquire mass, pre-sumably due to the oxidation in stages of the amino groups on787
Similar documents
View more...
Search Related
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks