sik tin Sats Sao nae Journal of The Electrochemical Society, 189 (5) 182-1187 2012) a The Elesothenial Sey Fluorescence-Signaling Aptasensor for ATP and PDGF Detection on Functionalized Diamond Surface A, Rahim Ruslinda,#™ Y, Ishiyama,* X, Wang,* T. Kobayashi," and H. Kawarada* “Deparment of Nanosclence and Nanoenginering, School of Advanced Selence and Engineering Wave Universi, Tokyo 169-8555, Japa Stnsatute of Nano Electronic Engineering, Universi! Malaysia Perlis, 01000 Kanga, Perl, Malaysia ‘An inated dlamond-based apaseasor is presented fr adenosine phosphate (ATP) and plate derived growth Factor (PDGE) ‘eteton. In hi work, a signa-off detection method was employed to provide a sense, slectve an reusable plato forthe PDGF and ATP, The detection i in this sty hat the ptasensor i Simple detect ako demonsts 62012 The Hlectochomical Society. [DOE 16.4 57/208 ‘0.2 nM and 0.2 M of PDGF and ATP were achieve respect sve for PDG ‘ofthe plaense ws contre by performing multiple cls, which shows tha ianond isa promising candidat boseaser applications. Moreover, this dtction system doesnot rogue ata es] Al eights reserved. Jy. lt was and ATP over tbe biomolecules. ln adliton, the etsy rial foe labo adit is unaffected by nonspecific Binding. “Manuscript submited November 28,2011; revised manussrip received February 17.2012. Published March 2, 2012. Paper 2688 presen tthe Moston, Massachusetts, Meeting the S To utilize an aptamer for biosensor application, the immobiliz tion ofthe aptamer to the sensor surface is considered as an important step in sensor design. The design challenges include many factors such 8s minimizing the non-specific binding, loss of affinity and inereas- ing the uoeessibility of the aptamer. Aptamers resolve their potential as sensors when they are covalently immobilized on a solid surface. AAptamers are shor, single-stranded oligonucleotides that have been selected for high affinity against a target protein, small molecule, or ‘even whole cells. Owing to their high sensitivity, selectivity temp ature stability, low cost, and reusability, aptamers have become the ideal recognition elements for varying biosensors. There are several possible immobilization strategies for DNA based molecules.” Anti= lbrombin aplamers were modified with an amino group atthe 3” end, ‘enabling covalent attachment to a glass surface via earbonylimida: zoe groups’ or aldehyde groups.’ Strategies for DNA immobilization in nucleie acid-based sensors with quartz erystal microbalance de tection of hybridization were developed several years go in various formats including biotin attachment and direct adsorption." These wore adapted to aptamer’ in biosensors with a biotin labeled ant-IgE aplamer immobilized on a streptavidin modified Ax.* Biotin or step lavidin immobilization staegies were also used in other aptamer biosensors."* However, the methods described above take several steps to immobilize the aptamer resulting in a higher risk for chemical ‘contamination and loss of specificity of the sensor Its therefore Vital t develop simple methods forthe attachment of aptamer to the surfaces for use in biosensors or diagnostics that involve minimal loss in funetionalty and non-specific interactions. Diamond has attracted significant attention because diamond is the best-known electrochemical uansduver owing to its chemical stabi ity low background current, wide potential window and outstanding biocompatibility.” ‘The diamond surface shows unique properties asitcan he modified with hydrogen, oxygen, uorine, carboxyl group and amino groups, which allows optimizing the propertis ofthe solid or electrolyte interface for biological materials such as enzyme,’ DNA!" and protein,”* Furthermore, covalent binding and clecto static adsorption are the most well known methods that have been used currently in diamond surfaces for DNA immobilization.” OF these methods, covalent binding onto several types of termination has heen used mainly, because these surfaces provide sites for amide or disulfide bonds, Howover, the oxygen terminated surfaces formed by dry oxida tion or wet chemical treatment provide several oxygen-related func tional groups such as Ketone, hydroxyl, and carboxyl groups, ‘The ‘method could subsequently limit the sueface coverage ofthe carhoxyl This was oct. Ostoier 9-14, 2011 group, which is crucial fr the immobilization of hiomolecules. How. ever in the case of amination, only NH bonds are expected to form ‘on the diamond surface due to their chemical structure." Further: e; the aminated diamond commonly keeps the structure fora ong time,’ The amide bonds formed by the interaction of carboxyl terminated biomolecules with an amine-terminated diamond surface are more stable than the commonly used Au-thiol surface bonds.” This is because the Au-thio surface shows rapid degradation owing to the easy hydrolysis of the thiol group under basic conditions." [Besides tha, Mluorine treatment have the hydrophobie behavior and the electronegatvity of fluorine contributes to the suppression of non specific hinding to @ great extent, which resulted in significantly in creasing the signal to noise ratio ofthe Muoreseence signal. Here, we report a signal-off method designated biosensor on aminated diamond surfaces focusing on. fuorescence-signaling aplasensor for adenosine triphosphate (ATP) and platelet-derived growth factor (PDGF) detection, ATP plays an important role asthe ‘major carrier of chemical enerey in regulation of cellular metabolism, biochemical pathways in cell physiology. I also has been used as an indicator for cell viaility and cell injury. Whilst, PDGF js related {o tumor growth as a potential cancer marker. It plays an important role in the regulation of cell growth and division. The efficiency fof aptasensor for PDGF-BB and ATP was investigated in the view of selectivity, sensitivity, stability and reusahility performance on functionalized diamond surface. Combining the bio-recognition elements of aptamers tothe solid sutfae lke diamond will represents a significant advancement, especially with respect to biomolecular reaction in vicinity of chemically functionalized carbon-based surface. Therefore, diamond as an active substrate interacting with aptamer immobilized on it has a potential that goes beyond a passive role asa substrate for anchoring biomolet Experimental Methods Direct amination on diamond surface— A polyerystalline dis mond was deposited by the mierowave plasma-assisted chemical va- por deposition method (MPCVD) on a silicon substrate (100) in 1% ‘methane gas diluted wih hydrogen gas for 4 h, After deposition, the diamond films were exposed to hydrogen plasma to realize hydro gen termination with a chamber pressure of $0 ‘Tort, For surface functionalizaion of diamond, partial surface amination of the hhydrogen-terminated diamond was performed by low-pressure mer. cry of ultraviolet light (UV) with the wavelength of 253.7 nm. A. gas was introduced into the reaetion chamber for $ min to purge the gases inside the chamber, then ammonia gas (99.9%) was fneoduced at 100 seem and the substrate itradiatod with UV light for 4h, The diamond surface was evaluated by X-ray photoelectronJournal of The Electrochemical Society, 189 (5) 182-J187 (2012) 1183 Lv iradiaven + ammosia gas $gsgd hens — tyarogen terminated darcond bi, NHL, NE, Ni, deen Padialy aninated diamond es Gold evaporator oy £vD. \ ip NHGNH, NNN, Pee F a re — oo — emcees pee by ICP-RIE ALLL igure 1. A micropater spectroscopy (XPS) analysis before and ater amination separately using an Ulvac 3300 (Ulvae-Phi, Kanagavea, Japan) with an anode source providing Al Ka radiation, Fabrication of diamond micropatiern.— A mieropatterns. were fabricated on aminated diamond using Au mask by photoithogrs phy. The micropatters on the partially aminated diamond suriace ‘were fabricated using the following steps: (1) deposition of the Aw mask by chemical vapor deposition (CVD), (2) prebaking at 80°C for min to remove adsorbed water molecules, (3) coating with photore sist by spin coating, (4) re-baking at 80°C for 20min, (5) paterning by aligner, (6) immersion in drying solution for 20 s, and (7) eth a the outside of mieropatiers by KI solution. For the cleaning of KI Solution and Au, the diamond surface of the etched Au was sonicated Urce times with 30 for § min, The remaining amino group was not ‘desired in KI solution after etching the Au mask, hocause the probe ‘oligonucleotides ean be immobilized within the existing amino group. To improve the signal-to-noise ratio, the area outside of the mieropal= tem was Muorinated by Cs plasma treatment (RIE-1DIiPH; Samco Intemational, Ine., Kyoto, Japan), because the nonspecific adsorption ‘of probe and target oligonucleotides can be minimized by the negative ‘charge of the luorine-lerminated diamond." A fabrication step of the ‘miropattern on functionalized diamond surface is shown ia Figure Chemicals and reagents.— The POGE-binding aptamer (i. 5 CAG GCT ACG GCA CGT AGA GCA TCA CCA TGA TCC 1G-3))." the complementary DNA of ATP aptamer (S-TGG_ ACC CCC TCA TAA CGC CIC CTE CCA-3'¥! mosified with COOH at the 5° end and the 27-mer ATP-binding aptamer (ie CyS-5'= GAG GAG GCG TTC TGA GGG GGT CCA-3) labeled with Cy at the S” end were synthesized and puritied by Sigma ‘Genosis Company (Hokkaido, Japan). The interelating dye solution (VOTO (1,1-(4,48,8-teramethyl-s,8- SSC and incubated at oom temperature for | hour. After that, the sample was rinsed using DI water and again the Nuorescence signal was measured. As for ATP. the ATP-binding aptamer was hybridized tothe complementary DNA, ‘of ATP aptamer (probe DNA) with the concentration of 1.0 1M in 2XSSC for 1 hat 25°C. The sequence is completely complementary to the probe DNA because the ATP-binding aptamer itself hears four pairs of complementary bases at its both ends (ic, S-EGG AAG. GAG GCG TIA TGA GGG GGT CCA-3)* and accordingly, its complementary DNA inherently has four pairs of complementary bases tie, STGG ACC COC TCA TAA'CGC CTC CTT CCA: 3)) at both of its ends, The affinity of ATP is not affected to the ‘uptamer that even overcomes Watson-Crick base pairs which has been demonstrated by Ving Lu etal. group. Then the fluorescence signalsist Journal of The Electrochemical Society, 189 (5) 182-1187 (2012) Sh sa aera? ca + h CC set we] oo) EL Sat maar 3h any (a) na as ‘agent aig enor Figure 2, Xaypotoolecton spectra of polserysallie damon surface. (a) High esolton scan of the Is poak ad) high esolation sean othe N Ts peak ane compared before un afer ect amination, was measured. Finally the ATP was introduced and incubated for Lh Fig. 2s the main carbon C 1s from diamond was detected a atypical 125°C followed by the Muorescence signal measurement. carbon peak position, which includes sp? and sp?, at 284.4 eV and 285.0 eV, respectively. The postions of the peaks at 285.5, 286.2 and 288.2 eV were identified and correspond to C-N, C-O and Results and Discussion bonds, respectively. The eoresponding ratio of NIC is 0.08. In ai- XPS analysis.— Two different kinds of diamond surface, be- ton, the existence ofthe amino group onthe diamond was eonfimed fore and after amination were evaluated hy XPS to determine the by the presence ofthe N speak after amination in Fig, 2. The dem amine groups generated onthe diamond surface by direct amination, sity of amino groups is evaluated from the N Is-to-C is peak-area Figure 2 shows the XPS spectra for C Is and N'ls area before and ratio but no from C-N to C-C peak-area ratio due to the very smal aller direct amination. Amino groups are supposed to exist tthe high- (0.2 €V) binging energy differences between C-C (sp?) and C-N, density regions of the diamond surface hy amination processes. The ‘The results show that the amino groups were successfully formed on simple reation of C-H bonds with ammonia gas undergoes a photo- diamond surface by direct amination, ‘chemical action one exposed to UV iradiation. This resulted inthe formation of €-NH, on diamond substrate, though steric limitations Fluorescence signal detection. The illustration diagram of the will imit amine group coverage onthe surface ta certain level." In aptasensing mechanisms of PDGF and ATP are shown in Figure (a) POGF-BB detection K- K- Probe aptamer lone addi POGE-BB (b) ATP detection ad removed ~ —~& Y ATP Complementary sang Hybedlzation of ATP-binaing aptamer WY ‘of probe aptamer labeled ATP-binding aptamer were conjugated ioATP Figure 3. A PDGE-BB and At sing mechanism on aminated-diamond apasenseJournal of The Electrochemical Society, 189 (5) 182-J187 (2012) os 07 06 os 08 03 02 on 0.0 ot 02 10 Relative Fluorescence decrease (a.u,) ie ibe a0 uss Gi ies 10h a0 aon PDGF concentration (nM) Figure 4 TOUM, respectively Here, we used the existing DNA aptamer for PDGF and ATP to test, the feasibility of signal-off strategy using the molecular light switel TOTO and labeled ATP-binding aptamer, respectively. A PDGF bind: Jing aptamer is reported to form a stem loop structure that can capture the intecalater uoreseent dye, resulting in significant fluorescence signal. As PDGI-BB is added to the PDGF-binding aptamer, the i= terealater dye dissociates due tothe change of the stem loop steuture in throo-way helix junctions. This results in a doctease in tuores ‘cence signal intensity indicating the presence of PDGE-BB daring the sensing detection method. As for ATP detection, the complementary Strand of aptamer was immobilized on aminated diamond surface us probe DNA. The labeled ATP-binding aptamer were hybridized into the sample and fluorescence signal was abscrved indicating the con formational change of duplex structure. When ATP s introduced into the sample, the labeled ATP-binding aptamer plays an important role {oconjugate with ATP and disintegrated from the probe DNA, which ‘was confirmed with the decreased in the fluorescence signal, These methods enabled accurate detection of PDGF and ATP existing in the sample with a Tow signal-to-noise background caused by physical sudsorption of both ATP andl PDGF themselves Aptasensor performance on sensitivity, selectivity and reusability Jor PDGF-BB detection. Figure shows a relationship between the relative fluorescence signal and PDGE-BB concentration. ‘The nor- ‘malized distributions of the PDGF concentration were ploted inthe range of 100 1M 10 100 nM. When the concentration of PDG-BB. The sensitivity of PDGF-BB aplasensr from range 14M to 100 nM, The concentrations of the probe aptamer an intrcalasr were 20 Mant decreased to 100M, the fluorescence signal intensity also decreased ‘ceordngly The volume of probe aptamer and interealater were eon Sant at 20 UM and 10 WM, respectively. The fluorescence signal intensity is found t be related with the effective number ofthe eon jugated structures, formed hy probe aptamer and PDGE-BB protein ‘as well as intercalaters with quite high affinity, The detection limit of| this aptasensor for POGE-BB was experimentally determined to be 10.2 nM based on signal-to-noise >3 while the limit of quantification (1.00) was determined to be 0.7 nM. This sensitivity is apparently better than tha previously reported for PDGE deteetion with different methods." The achievement ofthis detection Fmt will be sufficient tobe used for wide application in preliminary diagnosis cancer A selectivity of PDGF-BB aptasensor on diamond surface was investigated aguinst other biomolecules such as BSA, Calmodulis Glucose oxidase, Urease, ATP, and PDGF isoforms of PDGE-AB and PDGF-A. As shown in Fig. $8 only PDGE-BB caused a marked rc duction in uorescence signal, while all the other biomolecules failed to cause any significant reduction in fluorescence signal. The fuo rescence change of the aptamer with intercalater is highly selective due to the inherent specificity of the aptamer toward its target pro twin, However, the selectivity toward its isoforms, PDGE-AB revealed that a reduction in uorescence signal compared to PDGE-BB was ‘observed in about half of the signal intensity. This is because the sim ‘larity of amine acid sequence of PDGE-AB with PDGE-BB is only about 604. On the other hand, the PDGF-AA can also hinds tthe ‘uplamer but the affinity caused by PDGE-AA was clearly lower than 7, FD i $ sow copay wit) bet Bonclecls sich 7 as BSA, calmodulin, urease, glucose Bo. § ow: ‘oxidise and ioforis like PDGE-AB, 5 to fed POGEAA. Te concctntons of Eos : ML Sonntccs "0d. boleene were 5 5 sono. ToD nb. th) Reusable ofthe aplascnsor on Li PDGE-BB detection was observed for thr y= day ove veel . x tapasg 48 40 38 30 i | i j i AP oneeation Relative Fluorescence decrease (@.u,) 2 40) «BD ATP concentration (uM) 100 Figure 6. A response of apssensor for ATP concentration from 0.1 M10 10DaM, The inset shows the detection limit of ATP concentration rm (1 y.M 102 4M. The limit detection of ATP was down 100.2 AM, The concertos of the complementary stand of apamer and labeled ATP-binding apace ‘were 20 UM and I 4M respective PDGE-AB, Thus this aplasensor can distinguish isoforms with high selectivity To examine the reusability of aptasensor toward PDGE-BB on lireetly aminated diamond surface, we investigate the repetitive eycles involving the binding and denaturing of POGE-BB using 10% sodium ‘dodecylsulfate (SDS) treatment for 20 min in thee eyeles over three days, Fig, 5b indicates that the relative fluorescence is maintained when the binding-denataring eycles was performed in three cycles, ‘This result confirmed the reusability of the aptasensor, where the aptamer only bind selectively to PDGE-BB. Sensitivity, selecivty and reusability of aprasensor for ATP. derection.— The presence of ATP at diflerent concentration led to a \ifferent fluorescence signal intensity, depending on the amount of the released aptamer inthe solution, In order to evaluate the performance of the sensor, a series of ATP detection on aminated diamond surface was investigated from 0.1 jM to 100 wM. The difference in fuoreseence signal intensity was wsed to evaluate the fluorescence @) Rolative Fluorescence decrease (a...) ap uF oF Figure 7. sch as UTP, CTP and GI? The concentrations ofall he biomolecules were 1M. (5) The reusability of apasensr wis user over oa cycles i or Relative Fluorescence decrease (a.u.) Journal of The Electrochemical Society, 189 (5) 182-1187 (2012) signal response of ATP. As shown in Figure 6, the mduetion of fMuoreseence signal intensity is bigger with inereasing ATP concen trations. The sensor showed linear relationship berween the relative Auorescence decrease and ATP concentration ffor 0.1 wM 102.0 kM with the correlation voefliient of 0.986 (Figure 6 inset). The Timit of detection (LOD) and lower limits of quantification for ATP was experimentally determined to be 0.2 M and 0.7 41M, respectively, bused on a signal-to-noise rato of 3, which i lower than that inthe previous report. (On the other hands, the selectivity of aptamer toward ATP was very high as shown in Figure 9, We examined the selectivity of ATP and NTPsin lower concentration of | yM. lis found that this aptamer showed high specificity on ATP as compared with other related group ‘of ATP biomolecules such as NIPs (UTPs, CTPs and GPs). This js hecause these NTPs failed to cause any Significant signal. These results indicate that this aptasensor ean control the selectivity of ATP {in different kinds of concentrations by controlling the ATP-binding aptamer concentrations, Four repetitions of each standard ATP solution were carried out to evaluate the reproducibility and precision of the method, The re sls indicate that the aptasensor can be reused as shown in Figure 7, ‘which shows its potential as reusable biosensor, The higher intensity ‘ofthe fist eycle’s pre-hybridization was negligible as itis caused by physical adsorption ofthe aptamers onto the diamond sueface, These ‘Physical adsorption can be reduced easily by chemical functional: lization, such as oxidization or Nuorination, in the same way as we decreased the physical adsorption in our previous study!" The reusability and Mabilty of probe DNAs obtained are due to the strong covalent binding of the chemically stabile amino bonds introduced via photochemical amination, which is of great advantage on the sp” carbon based diamond surface, Biological samples.—To test the practicality of this present ‘method, the PDGF-BB has been applied to the potential interference such as bovine serum albumin samples directly, Two samples we twsed which are of undiluted bovine serum and of a contaminated Fidden sample of 50% BSA, As shown in Figure the feasibility of signal-ofF method can sill be observed in the PDGE-BB concentra tion range from 0.01 nM ~ 108) nM, Clearly, the sensitivity forthe setection with bovine serum albumin sample cannot be as high as that forthe pure one due to adsorption of the complex matrices and ‘non-target proteins svch as human serum albumin, Nevertholess, this signal-off method could still be used to detect the PDGE in biological samples for early diagnosis of clinical samples. However, the com plex matrices for ATP ate sil in progress for further investigation, @ 5 g g § ¢ "The selectivity and reusability of ATP on amined diamond surface was performed. (2) A comparison of selectivity on ATP with ther eat group wo dasJournal of The Electrochemical Society, 189 (5) 182-J187 (2012) ust » Undiluted serum = 50% BSA 050 045 040 038 030 025 020 045 010 095 Relative Fluorescence decrease (a.u.) 000 ° 20 0 60 80 PDGF-BB concentration (nM) Panthermore, the evaluation results of PDGP-BB in serum albumin ean give a model lor other biomolecules on diamond as a solid support. ‘Conclusions Inconclusion, the detection of PDGE and ATP on aptasensor-based aminated diamond surface has been performed successfully in terms of sensitivity, selectivity and reusability. This study provides clear ‘evidence that the apramer directed immobilization approach could be applied as a simple method to assemble sensors with high speciticity. The low signal-to-noise background of PDGF and ATP detection atest a high sensitivity sensor within a repeatable binding-denaturing ‘eycles and high selectivity against other biomolecules. In aptamer technology, simplicity and specificity are the important Teatures and thus, this finding holds a great potential fr future medieal diagnosis, Acknowledgments This work was supported by a grantin-Aid from GICOE Re- search from the Ministry of Education, Culture, Sports, Seience and Technology, Japan, and a grant-in-Aid for Fundamental Research A (23246069) from Japan Society for the Promotion of Science ISPS) tnd Marubun Research Promotion Foundation, References, 1. $1, Reape Cae Mo Chem 0), 1218 200 2 RA Pegntlo,RC Coorah AD. Biimgon, and GM. Hii, dn Sten 09198, 4. Caruso, E Rods, DF Furlong, K- Nika and ¥. Oat, #183139 2009, Figure 8. The sensitivity of ptasnsor was investigated in biological sample with the concentation of PDGF BB aissoved in fed square) united BSA. and (black squat) 50 BSA 100 ‘.Yanagshi, Shem, Tatsumi. Os 11 1925 95) Mins. Pare, H Wola, Pia, na? Chey PAID, A Le et834 291 2008, Ee Yan YZ 2, YA Zo Xi ad WL aS, 46, S892 ba i910, 01 (88. T. Knirbocke, Le Lancer, NR Ls MS ad RJ Hamers ie or SK} 1351 8 erie, 4,429 (2007), is zs ‘Wena Yanga Robe Her pi Pn, et U6), 3696 200) Ti Kara, asm 23 11248 000, " ‘ALR Rotini, 8 Tj, YY. Iiyama R.Bipington and H, Kava, dosnt, 20), 199 2010) me zi wie Chey 18, $2 Crane. H. Uncawa, H. Hata 1 Zabo, T. Fat, L Ohio, and Kral fps ay P39 C009, Xt fig. A Sen ht xm sab Wan, Chef 829 203, Y.tanX Lite Ahngs Pl Sian Maa Chm a (885008) Slate 3X Sat. Hou BM XH ag and Le Ln Tg. 2 Fang, a Bai ct Che, TL), 520 20. X Fang. Can Bek, nd W. Ta, nat Che 7, $725 200) Pine Fee 9, 1232 GO .