Gold nanoparticles (AuNPs) - Detection of Y-chromosome bearing bovine sperm using laser-generat

2. Literature

2.5 Gold nanoparticles (AuNPs)

Gold nanoparticles (AuNPs) have a size between 1 and 100 nm. They have unique versatile characteristics and have been investigated and used in many applications for technology and biotechnology, for example as therapeutic agents (BABAEI and GANJALIKHANI, 2014), drug delivery (BROWN et al., 2010, STUCHINSKAYA et al., 2011, BAKSHI, 2014), electronic conductors and catalysts (PITA et al., 2013, LI et al., 2014), inert bio-markers as replacement of fluorochromes, and toxic dyes (KLEIN et al., 2010, TORABI and LU, 2014).

Different aspects of AuNPs make them interesting for the scientific community: they are inert to cell function and easily synthesized by different methods; they are tunable and have unique optical and electronic properties. These characteristics are always due to the elemental compositions of the particles (DANIEL and ASTRUC, 2004, NEUMEISTER et al., 2014). Gold nanoparticles and nanotechnology in general are key components of future technologies in biotech industry, and in a wider range of

products from cancer detection systems (LONGO, 2014) to building artificial brains (MAYNARD, 2014).

The zeta potential is what mostly characterizes AuNPs and their optical properties when used as markers. Zeta potential refers to the electro-kinetic on the nanoparticle surface and is made of surface plasmons, oscillating electrons of the AuNPs. Surface plasmon resonance (SPR) leads to an optical absorption in the visible/infrared spectrum (BENETTI et al., 2013).

Interaction between AuNPs and light is strongly influenced by environment, size, and shape because of the change in the zeta potential. For example, for small (about 30nm) and monodisperse gold nanoparticles the SPR causes an absorption of light in the blue-green portion of the spectrum (~450 nm), while red light (~700 nm) is reflected, yielding a red color. As particle size increases, the wavelength of SPR related absorption shifts to longer and redder wavelengths. Red light is then absorbed, and blue light is reflected. The plasmon related absorption peak undergoes very large shifts in its wavelength, from 650 to 900 nm when differently dispersed in a medium or on a substrate. AuNPs potentially exhibit tremendous optical applications of detection if related to the absorbance (LI et al., 2014).

Aggregation is also a phenomenon that causes a shift in SPR. The aggregation of AuNPs of appropriate sizes induces interparticle surface plasmon coupling, resulting in a visible color change from red to blue (SAHA et al., 2012).

Fig .4: Example of gold nanoparticle bathocromic effect. A) Gold nanoparticles of same size and shape but differently agglomerated, reflect a different visible light. B) Different sized gold nanoparticles reflect different wavelengths of visible light.

AuNPs become molecular markers, when labeled to probes that guarantee specific binding and accumulation on target molecules (protein, DNA, drugs) causing the color change (WANG et al., 2014). The color change during AuNP aggregation provides a practical absorption-based colorimetric sensing for any molecular target that leads to AuNP agglomeration or dispersion (ROSI and MIRKIN, 2005, LIU et al., 2007, JIANG et al., 2010). Such sensing represents a valid alternative to photo bleaching problems related to fluorochromes, and a more suitable solution for biology excluding toxicity. Moreover, AuNPs can serve as excellent fluorescence quenchers for FRET-based assays (Fluorescence Resonance Energy Transfer) for sensing small organic molecules (CHEN and CHANG, 2004, HUANG and CHANG, 2006, JAIN et al., 2007). AuNPs provide non-toxic carriers for drug and gene delivery applications due to their characteristic promoting up take by tumor cells (GHOSH et al., 2008, WANG et al., 2014).

AuNPs cell internalization was obtained especially in cancer cells or tissue (GIBSON et al., 2007, SALMASO et al., 2009, TAYLOR et al., 2010, HAINFELD et al., 2014).

Uptake in tumor cells of LNA functionalized AuNPs was reported by Huo et al. (HUO et al., 2014). Microglial cells are also able to take up AuNPs, if these are labeled with cell penetrating peptides (CPPs) (HUTTER et al., 2010).

Gold nanoparticles are conventionally generated through chemical reduction methods (CRM) and several protocols are reported and reviewed (DANIEL and ASTRUC, 2004). Unfortunately, these procedures are known to be inefficient, since a significantly high excess of ligands and toxic residues remain in the AuNPs (ALKILANY and MURPHY, 2010).

AuNPs synthesized by pulsed laser ablation in liquid (Figure 5) is an alternative to conventional synthetic methods and it provides ligand-free NPs avoiding chemical residues (BARCIKOWSKI et al., 2007, BARCIKOWSKI and COMPAGNINI, 2013, REHBOCK et al., 2014). Laser generation of AuNPs also allows control of size and shape and their subsequent conjugation with organic molecules (REHBOCK et al., 2013). Pulsed laser systems irradiate solid state gold. Particle size can be tuned by variation of specific laser parameters (femtosecond, picosecond or nanosecond pulses). Functionalization with molecules like nucleic acids and proteins can be achieved in situ (direct addition of the functional agent to the medium prior to the laser process) or ex situ (the functional agent is mixed with the particles in a second synthesis step) (PETERSEN et al., 2009, PETERSEN et al., 2009).

Figure 5: Gold nanoparticles synthesis and conjugation. A) Process and course of laser-generated gold nanoparticles in water. B) Ex situ conjugation of AuNPs to TFO, separated synthesized TFO and AuNPs are diluted in a same tube. C) AuNP/TFO bio-conjugate.

Strategies that use DNA hybridization to control the placement of nanoparticles in one and two dimensions have been demonstrated (PINTO et al., 2005, NYKYPANCHUK et al., 2008, LI et al., 2014). Interaction of AuNPs and DNA was described to have a bi-exponential kinetic curve and reveal the presence of three kinetic steps. The first is a diffusion step with the formation of an unspecific precursor complex made of target DNA and AuNPs. The second step involves binding between hydrophilic groups and the DNA grooves and finally, the third step has been interpreted as a consequence of a conformational change of the first complex forming a more compacted form of DNA/AuNPs structure triggered by specific DNA hybridization (PRADO-GOTOR and GRUESO, 2011).

28 2.6 Expected insights

This research aims to find an innovative method to detect Y-chromosome bearing bovine sperm using AuNPs triggered by specific oligonucleotide hybridization.

The project started up considering flow cytometry sperm sorting issues, mainly represented by Hoechst dye 33342, which has a known potential risk of mutagenesis. A different signaling method may be found using different sensors.

Gold nanoparticles represent a valid, reliable, and innovative tool for molecular detection because they are inert, they show no photobleaching, they are easy to synthesize, and they are easy to be labeled with molecular probes guaranteeing target specificity. For instance AuNPs can be triggered by oligonucleotides to be used as sensor for gene targeting. The challenge in this thesis is to investigate oligonucleotides functionalized AuNPs, able to detect genes of highly condensed sperm DNA.

Moreover, it is expected to elucidate new aspects of biotechnology: (i) to understand if intermolecular triplexes are possible in a highly condensed chromatin configuration of the sperm nucleus; (ii) to investigate gold nanoparticle/bio-conjugate interactions within the bovine sperm chromatin; (iii) to provide assumptions on sperm chromatin structure and detection in bovine sperm for qualitative sorting; (iv) to validate the use of triplex hybridization with sperm chromatin.

29 3. Materials and Methods

3.1 Bovine Y-chromsome triplex target sites

Specific triplex target sites on bovine Y-chromosome were found using two software programmes. One is Triplexator (BUSKE et al., 2012) and the second one was written by Dr. D. Werner from University of Essen. Dr. Werner’s program was used to search for sequences and double checked with Triplexator. Both software programs were used to analyze the sequence of Bos taurus Y-chromosome deposited from GenBank accession no. CM001061.2. Ten sequences were taken for experimental validation through Southern blotting.

Triplexator identified triplex target sites in double-stranded sequences able to accommodate a third strand and also assesses the compatibility of potential TFO/TTS pairs according to the canonical triplex formation rules (BUSKE et al., 2012).

A list of TTS sequences was with the promt: >triplexator -l 15 -e 0 –m R–mrl 4 –mrp 4 –of 0 –o results.tts –ds ychrbull.

With such promt it was set up a research in the database for TFOs with characteristics: not smaller than 15 bases (-l 15), no error rate (-e 0), a TFO made of poly-pyrimidine sequences (-m R), with multiple repeats of 4 bases (-mrl 4 –mrp 4), avoiding off-targets (-of 0). List of TTS was made with the name of results.tts and analysing a FASTA file containing the entire Bos taurus Y-chromosome sequence (GenBank accession no. CM001061.2).

Triplex inspector is a different software suite able to calculate possible off-target sites (non-specific sequences that differ from the real target for about 5% of the nucleotides). Off target sites were analyzed for 20 sequences and listed in appendix, table 6 (BUSKE et al., 2013).

An additional BLAST analysis (The Basic Local Alignment Search Tool), which directly approximates alignments that optimize a measure of local similarity was also performed to check the sequence specificity on NCBI’s database (National Center for Biotechnology Information) (ALTSCHUL et al., 1990). On the webpage http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=OGP__9 913__10708 it is possible to check if the sequences listed by Triplexator belong to the bovine genome and if they are located on the sex chromosomes or autosomes.

3.2 Genomic DNA extraction from blood

Genomic DNA was isolated from white blood cells of female and male cattle. First, 10 mL of blood were diluted 1:1 in PBS, and centrifuged at 800 x g for 20 minutes. Pellet was washed 1x with 20mL of distilled water and vortexed for 1 minute to cause hemolysis . Thereafter 20 mL of 2x PBS were added to stop the hemolysis and centrifuged at 800 xg for 20 minutes. Hemolysis step was repeated at least 2 or 3 times until hemoglobin tracks disappeared. Cells were treated with a lysis buffer (50 mM Tris-HCl pH 8, 100 mM EDTA, 100 mM NaCl, 1% SDS) and 1mg/mL protein kinase. Each sample was incubated at 55 °C over night. The sample was centrifuged for 15 min at 20000 x g and the supernatant was mixed with 1 ml of saturated 6M

NaCl, and centrifuged for 15 min at 20000 x g. The pellet was washed with 1 ml of ethanol 100% and centrifuged for 15 min at 20000 x g. A second washing step was performed diluting the pellet in 1ml of 70% ethanol at 37°C for 2 hours on a thermo-shaker. After centrifugation for 15 min at 20000 x g the pellet, containing the DNA, was dried in air and diluted in distilled water. gDNA concentration and purity were measured by spectrophotometry at 260 nm wavelength (NanoDrop ® ND-1000 Spectrophotometer).

3.3 Southern blot analysis

Genomic DNA from male and female cattle were digested as follows: 400ng of gDNA were incubated with 5 U of PstI (New England Biolabs, INC.), 0.1x BSA, 1x Pst buffer (New England Biolabs, INC.) for 2 hours at 37°C.

Southern blot was performed according to Current Protocols in Molecular Biology, 2003 (Unit 2.9A Southern Blotting) (MARCADET et al., 1989, SOUTHERN, 2006).

Electrophoresis in 0.8% agarose (TAE buffer) was performed of 1µg and 4µg respectively of bovine male and female gDNA for ~2 hours at 100V. The gel was first treated with 0.25M HCl for 10 minutes and washed with distilled water. A second treatment with denaturation buffer (1,5M NaCl and 0.5M NaOH) was made for DNA denaturation for 20 minutes, and washed with distilled water. The gel was finally washed for 20 minutes in neutralization buffer (0.5M of TRIS HCl pH 7 and 1.5M of NaCl) to neutralize the denaturation reaction. Gel was finally incubated with 20 x SSC for 10 minutes.

The blotting was performed via upward capillary transfer of DNA from agarose gel onto a nylon membrane, using a 20x SSC buffer. DNA immobilization was achieved by UV irradiation (120x10³ µJoule for 10 sec). The membrane was incubated at 42

°C for 16 hours with digoxigenine (DIG) labeled probes in hybridization buffer (DIG Easy Hybridization buffer by Roche) at concentration of 25 ng/ml. Probes for hybridization were synthesized by Eurofins MWG Operon (Eiserberg – Germany).

Washing steps after hybridization were made with high stringency to increase binding affinity. The digoxigenin-based detection system (Boehringer Mannheim) was assessed by incubating the membrane for 10 minutes in detection buffer (100ml 1M Tris HCl pH 9 and 33.3ml 3M NaCl) and CDP-Star® Chemiluminescent Substrate at a concentration of 0.25 mM. Membrane was then ready for chemiluminescence detection. Chemiluminescence and fluorescence detection were made using FUSION® image system acquisition.

3.4 Triplex melting curve analysis

Melting temperature curve is an analysis of the dissociation-characteristics of double-stranded DNA during heating and the same principle is valid for triplex double-stranded DNA analysis. The measurement is made using spectrophotometry, detecting the relationship between DNA absorbance and its structure. The physical principle is based on the fact that DNA structure has a lower absorbance (a value that correlates the light absorbed by a chemical substance) when in triplex formation. Rising the temperature, the triplex DNA structure melts and the third strand detaches from the target dsDNA corresponding to a different absorbance. A further increase of the

temperature will melt the dsDNA. Two variations of the absorbance at two different temperatures were recorded, one for triplex melting and a second for the duplex melting.

dsDNA target (listed in table 1) were designed in a way that 2 molecules of the same TFO could hybridize to one molecule of target dsDNA, as represented in figures 6.

Figure 6: Melting temperature principle of analysis. A) Structure of target dsDNA in black and TFO B) principle of melting temperature analysis, rising the temperature a first melting point is recorded due to melting of triplex structure. A second melting point is registered at higher temperature due to dsDNA melting. Absorbance is directly proportional to melting temperature.

Table 1: Target dsDNA sequences for each TFO

Name Sequence

Target Seq1 Fw 5’AGAAAGGAAGAAAAGGAAAGAGAAAGGAAGAAAAGGAAAG Target Seq1 Rw 5’CTTTCCTTTTCTTCCTTTCTCTTTCCTTTTCTTCCTTTCT Target Seq2 Fw 5’AAGGGAAAGGAAGGAAGAGAAGGGAAAGGAAGGAAGAG Target Seq2 Rw 5’CTCTTCCTTCCTTTCCCTTCTCTTCCTTCCTTTCCCTT

Target Seq3 Fw 5’GAGGGAGGGAAAAGGGAGGAAGGAGGGAGGGAAAAGGGAGGAAG Target Seq3 Rw 5’CTTCCTCCCTTTTCCCTCCCTCCTTCCTCCCTTTTCCCTCCCTC

ssDNA for dsDNA target were formed by incubating each complementary probe at a concentration of 100pmol in water solution. The sample was warmed up to 95 °C and slowly cooled down.

To assess the melting temperature experiment, target dsDNA was first synthesized.

Complementary ssDNA target oligonucleotides were ordered from Eurofins MWG Operon (Eiserberg Germany). Complementary probes were diluted in distilled water at equimolar concentration (100pmol/µL), warmed up to 95°C for 10 minutes, and slowly cooled down to RT in order to allow a dsDNA formation. Samples were stored at 4 or -20 °C.

Melting point was measured after incubation of 1µM dsDNA and 1µM TFO in 1x PBS pH 7 at 15°C and warmed up to 80°C. Measurements were made at a wavelength of 260 nm. Analyses were performed by University of Duisburg-Essen and Center for Nanointegration Duisburg-Essen (CENIDE).

3.5 Triplex electrophoresis mobility shift assay (EMSA)

Electrophoresis mobility shift assay is a method used to validate probes triplex binding under electric field that shifts the localization of the free unbounded TFO from the hybridized ones (VASQUEZ et al., 2001, HELLMAN and FRIED, 2007).

To assess the EMSA, 1µg of male and female genomic DNA was isolated from blood and PstI digested (chapter 3.2, pp 30). gDNA was then incubated with 50 ng of DIG labeled TFO1, 2, and 3 in 1x PBS buffer pH 7 at different concentrations of MgCl2 (5, 10, and 20mM) at 19 °C for 1h. After incubation the samples were rapidly chilled on ice. A mobility electrophoresis in 0.8% agarose gel (TAE buffer) was performed at a RT of 5 °C. After electrophoresis, the gel was kept in 20x SSC for 10 minutes.

A blot was performed of the DNA/TFO sample after electrophoresis via upward capillary transfer of DNA from agarose gel onto a nylon membrane, using a 20x SSC buffer. DNA immobilization was achieved by UV irradiation (120x10³ µJoule for 10 sec). As only TFOs were labeled to DIG, detection of DIG on the membrane was equivalent to detection of the triplex hybridization.

3.6 Sorted sperm nuclear matrix isolation

Bovine sperm were sorted as described by Rath et al. (RATH et al., 2009). More than 90% enrichment was achieved and purity validation of sorted samples was performed by reanalysis and by fluorescence in situ hybridization (FISH) (KAWARASAKI et al., 1998, RATH et al., 2009). Sperm were diluted in 10 mM

Tris-36

HCl pH 7, 1mM EDTA. Sonication was performed at frequencies higher than 20 kHz to disrupt heads from tails and a density gradient Percoll (Sigma Percoll P-1644) centrifugation separated respective sub-fractions. A falcon tube was prepared with 3 phases of differently concentrated Percoll: 90% Percoll on the bottom of the tube, 67,5% Percoll gently charged on it and at last a 45% Percoll was charged on top. 1 mL of sonicated sperm sample was gently loaded over the 45% Percoll. Tube was centrifuged for 850 xg for 20 minutes. Percoll gradient centrifugation separated the sample with heads on the pellet and tails between phases of 65% and 90%. The procedure was repeated once.

Sperm heads were treated for de-membranization with 10% TritonX-100 in a buffer made of 10 mM Tris-HCl pH 7, 1 mM EDTA and 250 mM sucrose (KURODA and PORTER, 1987). Washing steps were performed by centrifugating the samples at 20000 x g for 5 minutes and resuspending it in the 10 mM Tris-HCl pH 7, 1 mM EDTA and 250 mM sucrose . At last sperm nuclear matrix were diluted in distilled water.

To confirm that membranes were mostly dissolved without a loss of nuclear structure, a Neil red staining for phospholipids was assessed as follows: A Nile red stock solution was prepared (500 µg/mL) in acetone and diluted with 75% aqueous glycerol to a concentration of 2.5 µg/mL. One drop of Neil red was used on sperm and sperm nuclear matrix was fixed on a slide. Neil red staining has an excitation wavelength between 515-530 nm and an emission wavelength between 526 and 605 nm (DIANE L. PECHT and STEPHEN K. DURHAM, 2007).

A control group of normal sperm not subjected to demembranization was also stained with the same procedure. Images of demembranized sperm nuclear matrix and normal spermatozoa were evaluated under a confocal microscope (Figure 7).

Light microscopical visualization was performed using an Axioplan 200 and a confocal imaging system LSM510 (Carl Zeiss MicroImaging GmbH, Jena, Germany) within the spectrum of visible light. A (Helium-Neon-Red) Helium-Neon-green laser of 543 nm was used to excite the Nile Red staining with emission detection from 560 nm.

Figure 7: Confocal microscopy images of bovine sperm nuclear matrix isolation. A) Normal bovine sperm stained with Neil red; B) Same sample of bovine sperm demembranized and stained also with Neil red.

3.7 Triplex fluorescence in situ hybridization (T-FISH)

Microscope slides of sorted bovine sperm nuclear matrix were prepared by letting a drop of sample suspension fall from a height of about 20 cm on the center of the slide. Once dried, the slides were incubated at 19 °C for 2 hours in triplex hybridization buffer (1x PBS pH 7 and 10 mM MgCl2 and 1 mM of fluorescent labeled

TFO probes). Chemical hybridization is based on Hoogsteen binding between the TFOs and the sperm nuclear matrix.

A single washing step was assessed using hybridization buffer (1x PBS pH 7 and 10 mM MgCl2) at 19 °C. Finally slides were dehydrated with ascending ethanol series and mounted with Vectashield ® mounting media. Slides were stored at 4°C or immediately analyzed under a fluorescence microscope (Olympus BX60). In total 200 observations were made.

A fluorescent duplex in situ hybridization was made as control for each TFO to have a correct interpretation of the hybridization based on the presumption that hybridization triplex had to have the same location as a hybridization duplex.

First, TFO1, 2 and 3 were used separately, on a second step, all three probes were used together to analyze a possible better outcome. A fluorescent duplex in situ hybridization was made as control for each TFO to have a correct interpretation of the hybridization based on the presumption that hybridization triplex were to have the same location of a hybridization duplex. Since the result of the assay was a YES/NO hybridization, a Chi square analysis of 200 observations was performed.

Probes used for triplex fluorescent in situ hybridization were:

TFO1: 5'-YXYTYXXYTXYTYTXXYTYX-Alexa488 TFO2: 5'-TYXXXYTYXXYTXXYTXYX-Alexa488 TFO3: 5'-XYXXXYXXXYTTYXXXYXXYTX-Alexa488

39 3.8 Fluorescence in situ hybridization (FISH)

Fluorescent in situ hybridization is a common method used to identify specific gene sequences on the sperm heads based on a Watson and Crick chemical hybridization (RENS et al., 2001).

Slides with bovine sorted sperm were prepared by letting a drop fall from about 10 cm above on microscopes slides. Samples were dried for about 30 minutes at 37 °C.

Slides were treated for de-condensation with 3M NaOH for 3 minutes, rapidly washed twice in H2O and dehydrated with ascending ethanol series (70%, 90%, and 100%).

Slides were incubated at 37°C for 2 hours in hybridization solution buffer previously prepared (2x SSC, 30% formamide and 1mM of fluorescent labeled probe) (SILAHTAROGLU et al., 2007).

First washing step was made using 0.1x SSC tween 1% at 41°C for 5 minutes and in 0.1x SSC for 5 minutes at RT. Finally, slides were dehydrated with ascending ethanol

Im Dokument Detection of Y-chromosome bearing bovine sperm using laser-generated gold nanoparticle bio-conjugates (Seite 23-0)