ap-5-11.dvi Acta Polytechnica Vol. 51 No. 5/2011 Optical Detection of Charged Biomolecules: Towards Novel Drug Delivery Systems V. Petráková Abstract This paper presents work done on developing optically-traceable intracellular nanodiamond sensors, where the photolu- minescence can be changed by a biomolecular attachment/delivery event. Their high biocompatibility, small size and stable luminescence from their color centers make nanodiamond (ND) particles an attractive alternative to molecular dyes for drug-delivery and cell-imaging applications. In our work, we study how surface modification of ND can change the color of ND luminescence (PL). This method can be used as a novel detection tool for remotemonitoring of chemical processes in biological systems. Recently, we showed that PL can be driven by atomic functionalization, leading to a change in the color of ND luminescence from red (oxidized ND) to orange (hydrogenated ND). In this work, we show howPL of NDchanges similarly when interactingwith positively and negatively chargedmolecules. The effect is demon- strated on fluorinated ND, where the high dipole moment of the C-F bond is favorable for the formation of non-covalent bonds with charged molecules. We model this effect using electrical potential changes at the diamond surface. The final aim of thework is to develop a “smart” optically traceable drug carrier, where the delivery event is optically detectable. Keywords: nanodiamond, drug delivery, nitrogen-vacancy center, luminescence properties, charged molecules. 1 Introduction Fluorescent cellular biomarkers play an essential role in biologyandmedicine for in-vitro and in-vivo imag- ing in living cells. Luminescent nanodiamond (ND) has recently been suggested as a novel opticalmarker for cellular imaging [1,2]. ND offers advantages over classical fluorescent markers used for in vivo and in vitro imaging in living cells. It offers a cellular de- livery combined with strong and stable photolumi- nescence (PL) originating from the nitrogen-vacancy (NV) or other lattice point defects. ND is biocom- patible, and its surface can easily be terminatedwith various groups and additionally functionalized with biomolecules [3,4], making ND a suitable carrier for cellular targeting or drug delivery. In this work we showamethod for changing the PLproperties ofND in a biological environment by surface termination- induced changes leading to electric field development close to the ND surface. The NV center is observed in two charge states, negative and neutral, each with different PL prop- erties [5]. A negatively charged NV center (NV−) emits around 637 nm and a neutral center (NV0) emits around 575 nm. Under standard conditions, both states are observed, but NV− related lumines- cence is more intense. A single NV center can exist in both states [6], depending on its surroundings. In this work we demonstrate the influence of the pres- ence of charged molecules (biopolymers) on changes in the occupation of NV− and NV0 states in fluo- rinated ND, with NV− quenching upon interaction with positively charged polymers. The high dipole moment of F-terminated diamond attracts positively charged molecules, leading to the creation of a hole accumulation layer and therefore a charge transfer from the diamond to the surrounding polymer, i.e. an upward surface band bending. We use these ef- fects to induce changes in the occupation of NV0/− centers lying in the band-bending zone. We demonstrate these effects on NV centers in high-pressure high-temperature ND of 20–50 nm in size produced by irradiation and annealing. The re- sults are supported by theoretical modeling of the density of the state distribution for various surface interactions. The effect is compared with oxidized ND. 2 Experimental The effects presented in this work were studied on HPHT type Ib ND 40–50 nm in size, sourced from Microdiamant AG, Switzerland (size selected from commercial product MSY 0–0.05 GAF) containing about 100–120 ppm nitrogen (Figure 1). Fig. 1: AFM picture of the ND particles 89 Acta Polytechnica Vol. 51 No. 5/2011 Acommercial solution ofND was lyophilized and heated in air at 425◦C for 5 h to remove any sp2 carbon-containing layer. The resulting pale grey powder was dispersed in water and deposited in the formof a thin filmona target backing (10mg·cm−2) for ion implantation. TheNDwas then irradiatedus- ing an external proton beam produced by a U-120M isochronous cyclotron. The angle of the target back- ing with respect to the beamdirectionwas 10◦. The fluency of the delivered beam was 9.2 · 1015 cm−2, beam energy 5.4MeVand beamcurrent 0.6 μA.The irradiated ND was thermally annealed in a vacuum at 710◦ C for 2 h to create NV centers by trapping the vacancies next to the nitrogen atom. NDs were then oxidized in a mixture of concentrated H2SO4- HNO3 (9 : 1, vol/vol) at 80 ◦C for 7 days. The reaction mixture was diluted with deionized water, the NDs were separated by centrifugation and was subsequently washed with 0.1 M NaOH, 0.1 M HCl, and finally washed three times with water. The so- lution was lyophilized, providing highly fluorescent NDs in the form of a stable colloidalmonodispersion in water, as confirmed by AFM and DLS (final size 20–25 nm). The colloidal dispersion was stable after 2months, with no sedimentation. After themeasure- ment, the quartz plate with ND grains was exposed to microwave-excited hydrogen plasma for 30 minu- tes at a temperature of 500◦C and at pressure of 1 mbar to produce anH-terminated surface. Finally, the H terminated samples were fluorinated by ionic fluorination in amixture with an aqueous solution of hydrogen fluoride and fluorine gas. After saturation of the solution, the suspension reacts under pressure for 2 days. Raman and PL (514 nm) spectra were recorded using a Renishaw InVia Raman Microscope. Spec- tra were taken at room temperature. All spectra were normalized to the diamond Raman peak. The AFMmeasurementswereperformed in tappingmode (111kHz)withanNTEGRAPrimaNTMDTsystem equipped with a soft HA NC etalon tip. Polymers were chosen as positively charged molecules: poly diallyldimethyl ammonium chloride (PDADMAC) and polyallylamine (PAA.HCl). Poly- acrylic acid sodium salt (PANa) and polystyrene sulfonic acid sodium salt (PSSNa) were used as negatively-chargedmolecules. 3 Results 3.1 Comparison of PL on variously terminated ND The PL of NV centers is sensitive to the surface ter- mination, as shown in our previous work [7]. How- ever, the effect of surface fluorination has not been described yet. Figure 2a shows the PL spectra of fluorinated, oxidized and hydrogenated ND, where we clearly see the difference in the NV−/NV0 ratio, where the NV− luminescence is most pronounced in fluorinated ND. Fig. 2: Comparison of PL of variously treated ND. a) PL spectra takenat 514nmexcitation in a liquid colloid solu- tion showing increased NV− related luminescence for flu- orinatedND in comparison to oxidized and hydrogenated ND. b) Counted NV−/NV0 ratio of treated ND. The ra- tiowas counted from theNV− andNV0 zero phonon line (ZPL) — gray line in Figure 2a 3.2 Interaction with charged polymers Inamixture of nanoparticleswith chargedmolecules, non-covalent bonds are always formed between the chargedmolecule and the nanoparticle. The strength of thebonddiffers, anddependsonmany factors, e.g. 90 Acta Polytechnica Vol. 51 No. 5/2011 the ability to formhydrogenbondsor the sizeandpo- larity of the surface dipole moment of the nanopar- ticle. If the charged molecule is strongly attracted, charge transfer can occur, depending on the HOMO and LUMO energetic levels of the nanoparticle and the chemical potential of the molecule. Fig. 3: The negative and positive electric field formed in the close surface proximity of ND after the interaction with charged polymers Fig. 4: The PL spectra of fluorinated ND on interacting with the negative and positive electric field formed in the close surface proximity of ND after the interaction with charged polymers Four different types of polymers were chosen for this experiment (see Experimental for details). The polymer size did not exceed the size of ND, favoring the creation of stronger bonds. The colloidal solu- tion of ND was mixed with polymers in 10× higher concentration, enabling saturationof the surfacewith polymers. The interactionbetweenpolymersandND is schematically shown in Figure 3. Figure 4a shows the changes in the PL spectra of fluorinated ND when interacting with charged poly- mers. Figure 4b shows thePLof the interactionwith the same charged polymers, but using oxidized ND. The luminescenceof theNV-centers clearlydecreased on interacting with positively charged molecules, while after adding negatively charged polymers the luminescence was restored to the original level. When comparing the effects observed on fluori- nated ND with the effects on oxidized ND, we find that the effect is much stronger for fluorinated ND. This difference could be due to the different prop- erties of the carbon-fluor (C-F) and carbon-oxygen (C-O) bond. The electron affinity of the C-F bond is much higher (1.45) than the affinity of the C-Obond (0.9). This leads to stronger attraction of positively charged polymers to the fluorinated surface. How- ever, concerning the hydrogen bond, a fluorinated surface can be only an acceptor of a hydrogen bond, oppose to the oxidized surface (containing carboxyl, carbonyl, hydroxyl or lactone groups) can be both donor and acceptor of the hydrogen bond. 3.3 In-vivo imaging in chicken embryos Fluorinated NDs were used for in-vivo luminescence imaging in chicken embryos. The results (Figure 5) showan important fact that the intensity of the lumi- nescence from fluorinatedND is strong enough to be detected in a commercially available confocal micro- scope used for standard luminescence imaging. The method is therefore suitable for optically traceable drug delivery systems. 4 Modeling the effect To explain the observed effect, we modeled the ener- getic balance near the surface by numerical solution of the Poisson equation using the Boltzmann distri- bution. We can write for the depth (x) dependent total space charge density ρ(x): ρ(x)= eNV exp ( − (EF − EV )x kT ) , (1) where NV is the temperature dependent effective density of states at EV BM, NV = 2.7 · 1019 cm−3 at room temperature, e is elementary charge, k is Boltzmann constant and T is thermodynamic tem- 91 Acta Polytechnica Vol. 51 No. 5/2011 Fig. 5: Luminescence confocal image of fluorinatedND in a chicken embryo. NDvisible as green dots in the picture Fig. 6: DOS calculations of surface band bending for two different situations: a) fluorinated ND, b) oxidized ND. The unoccupied (dark) state where luminescence of the NV- centers cannot occur is larger for the fluorinated sur- face than for the oxidized surface perature. EF and EV are the energetic levels of the Fermi and valence band. d2(EF − EV )x dx2 = e εε0 ρ(x) (2) where εε0 is the permittivity of the material. ThePoissonequationwas solvednumericallywith boundary conditions (EF − EV )0 = kT ln ( NV p0 ) (3) p0 = e (NA · ζ) (4) p0 is the total unscreened positive charge at x = 0 from (1), and NA is the density of the surface ac- ceptors. The density of the surface acceptors was calculated by solving the Boltzmann-Poisson equa- tion, taking into account the different electron affin- ity values described above. The electron affinity was introduced into the model as parameter ζ. In our calculations, ζ was set to 1.4 and 0.9. The results of mathematical modeling are shown in Figure 6. The modeling clearly explains the re- duced effect. 5 Conclusions The luminescent properties of NV defects engi- neered in HPHT ND when interacting with charged molecules have been studied. It was found that the luminescence of NV centers is sensitive to surface treatment. The NV- luminescence fell significantly after hydrogenation of the surface and increased af- ter fluorination, in comparison with the standardly usedoxidized surface. Additionally, itwas found that the PL of fluorinated ND can be strongly influenced by the presence of charged molecules. This can be further used for in-vivo optical detection of charged molecules in cells/smart drug delivery systems. The observed effects have been explained by numerical modeling. The final result of this study was an in- vivo application of luminescenceND in a chicken em- bryo, showing the detectability of luminescence ND in a standard confocal microscope. Acknowledgement Special thanks to author’s supervisor, Prof. Miloš Nesládek, Hasselt University, Belgium for leading the research project, to Petr Cı́gler and Miroslav Ledvina for their help with surface modifications, to FrantǐsekFendrych andAndrewTaylor for their help concerning the properties and characteristics of dia- mond and for managing the research project, and finally to Jan Štursa and Jan Kučka for the oppor- tunity to irradiate ND particles in the cyclotron at UJV Rež. The author acknowledges financial sup- port from the Academy of Sciences of the Czech Republic (grants KAN200100801,KAN301370701& KAN400480701), the European R&D projects (FP7 ITN Grant No. 238201 – MATCON, No. 245122 DINAMO and COST MP0901 – LD 11076 and LD11078), and MSM6840770012 “Transdisciplinary Research in the Field of Biomedical Engineering II”. 92 Acta Polytechnica Vol. 51 No. 5/2011 References [1] Ho, D.: Beyond the Sparkle: The Impact of Nanodiamonds as Biolabeling and Therapeu- tic Agents, ACS Nano, 2009, vol. 3, no. 12, p. 3825–3829. [2] Fu, C. C., Lee, H. Y., Chen, K., Lim, T. S., Wu, H. Y., Lin, P. K., Wei, P. K., Tsao, P. H., Chang, H. C., Fann, W.: Characterization and application of single fluorescent nanodiamonds as cellular biomarkers. Proc. Natl. Acad. Sci. USA 2007, vol. 104, no. 3, p. 727–732. [3] Liu, K. K., Cheng, C. L., Chang, C. C., Chao, J. I.: Biocompatible and detectable car- boxylated nanodiamond on human cell, Nan- otechnology, 2007, vol. 18, p. 325102. [4] Kreuger, A.: The structure and reactivity of nanoscale diamond, J. Materials and Chemistry, 2008, vol. 18, no. 13, p. 1485–1492. [5] Davies, G., Hamer, M. F.: Optical studies of the 1.945 eVvibronicband indiamond,Proc. R. Soc. Lond. A., 1976, vol. 348, p. 285–298. [6] Ialoubovskii, K., Adriaenssens, G. J., Nes- ladek, M.: Photochromism of vacancy-related centres in diamond, J. Phys.: Cond. Matter, 2000, vol. 12, p. 189–199. [7] Petráková, V., Nesládek, M., et. al.: Lumines- cence of nanodiamond driven by atomic func- tionalization: Towards novel detection principles, submitted to Adv. Func. Mater., 2011. About the author Vladimı́ra Petráková was born in Prague, Czech Republic. She graduated with a master degree from the Faculty of Biomedical Engineering, Czech Tech- nical University (FBE, CTU) in June 2009. In Oc- tober 2009 she joined the Laboratory of Materials forNanosystems andBiointerfaces at the Institute of Physics,AcademyofSciences. Currently she is in the secondyearof herdoctoral studies atFBE,CTU.She received twice Josef Hlávka prize for the Best Stu- dents and Graduates (2007, 2009), and received the Dean’s prize for an excellent bachelor thesis in 2007. Her professional interests are luminescence centers in nanodiamond particles, high-resolution optical sys- tems, surface chemistry, biosensors, and also neural circuits and data analysis. In 2010 she received a Young Investigator Award for the best oral presen- tation in the European Diamond Conference in Bu- dapest, Hungary and also at the MRS Fall Meeting at theBoston,USA forherworkdescribing themech- anism and control of switching between the neutral and negative charge state of the NV center in dia- mond. Other interests are family, backpacking and music. Vladimı́ra Petráková E-mail: vladimira.petrakova@fbmi.cvut.cz Faculty of Biomedical Engineering Czech Technical University in Prague Sitna Sq. 3105, 272 01 Kladno, Czech Republic Institute of Physics AS CR Na Slovance 5, 185 00 Prague 8, Czech Republic 93