Oligonucleotides containing biotin functionality were successfully labelled with a streptavidin nanogold conjugate and subsequently separated and analysed by High Performance Liquid Chromatography–Inductively Coupled Plasma–Mass Spectrometry (HPLC-ICP-MS)

Barry Sharp (1249158)

Samantha Louise Kerr (7165235)

Chemical Communications

English

Loughborough University Institutional Repository

   This item was submitted to Loughborough’s Institutional Repository by the author and is made available under the following Creative Commons Licence conditions.      For the full text of this licence, please go to: http://creativecommons.org/licenses/by-nc-nd/2.5/  CREATED USING THE RSC COMMUNICATION TEMPLATE (VER. 2.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS COMMUNICATION www.rsc.org/[journal] | [journal name] This journal is © The Royal Society of Chemistry [year] [journal], [year], [vol], 00–00  |  1 Nano-particle labelling of nucleic acids for enhanced detection by inductively-coupled plasma mass spectrometry (ICP-MS) Samantha Louise Kerr and Barry Sharp* Receipt/Acceptance Data [DO NOT ALTER/DELETE THIS TEXT] Publication data [DO NOT ALTER/DELETE THIS TEXT] 5 DOI: 10.1039/b000000x [DO NOT ALTER/DELETE THIS TEXT] Oligonucleotides containing biotin functionality were successfully labelled with a streptavidin nanogold conjugate and subsequently separated and analysed by High Performance Liquid Chromatography–Inductively Coupled Plasma–Mass 10 Spectrometry (HPLC-ICP-MS). Inductively coupled plasma - mass spectrometry (ICP-MS) is the most sensitive and versatile analytical technique for providing elemental and isotopic information. Most elements in the periodic table can be ionised in the ICP source including biologically 15 important elements such as P and S and as a result, ICP-MS has been increasingly employed in the analysis of nucleic acids and proteins.1-3 ICP-MS provides complementary information to conventional organic MS and has many additional advantages.2, 4, 5 Firstly, ICP-MS offers very low limits of detection, which 20 generally range from pg/l - µg/l depending on the analyte. Calibration and quantification are much easier and require only inorganic elemental standard solutions. In addition, because ICP-MS measures the total concentration of an element, regardless of chemical form, it facilitates mass balance calculations, which are 25 vital for establishing analyte recovery and method validation. Although the ICP is a hard ionisation source, molecular information can be obtained, for example by measuring P/S ratios in proteins,  or by employing it as a selective detector coupled to separation techniques. 30  Despite the advantages of ICP-MS, P and S are problematic elements because they have high first ionisation potentials (10.5 eV and 10.4 eV respectively), which results in incomplete ionisation (~ 35% and 15% for P and S respectively)6, and they suffer from polyatomic interferences e.g. 14N16O1H, 16O2 and 14N18O.7, 8 Thus 35 whilst metallic elements can readily be detected at pg/l levels, the detection limits for these elements are much higher. The collision reaction cell method converts the analytes P and S to their oxides at m/z 47 and 48 yielding detection limits of 1 and 1.5 µg/l for 31P and 32S respectively.9 For a sector instrument, running at 4000 40 resolution, the corresponding values are 50 ng/l for 31P and 10 ng/l for 32S.9 These problems can be avoided and the biomolecule signal enhanced if it is labelled with a metallic element or a metal nanoparticle which is measured instead of P or S.10-11 The concept of elemental labelling within the field of atomic spectrometry has 45 been pioneered by Tanner et al. 8, 10, however, this area of research is still novel and continually developing.   Gold nanoparticles containing streptavidin functionality have been employed in this communication to label biotinylated DNA. Avidin proteins bind strongly to biotin with assosication constants 50 in the region of 1015 M-1, 12 in what is known as the strongest non-covalent interaction. This property makes these two molecules ideal linkers for biomolecule labelling. The limits of detection for Au are much lower than those of P and S due to an absence of interferences and a lower first ionisation potential. In addition, the 55 biomolecule signal is further enhanced since each nanoparticle contains approximately 80 gold atoms. The ICP-MS analysis of Au labelled DNA has been reported elsewhere, 13 however, the method employed DNA hybridised with a peptide sequence. Gold labelled secondary antibodies were used to label a monoclonal antibody 60 which was located at the peptide site on the hybrid biomolecule.13 The method detailed in this communication is site specific but not base sequence specific or dependent upon peptide sequences. It  is therefore generic and can be applied to any nucleic acid that is biotinylated. In addition, this method can be applied to small 65 nucleic acids, such as dinucleotides which are produced in the post labelling assay, which are not suitable for PCR amplification.14   For quantitative analysis of the Au labelled oligonucleotides, it is essential to know how many gold atoms have been bound and where.11 To satisfy this criteria, site specific labelling is required, 70 which was achieved by employing DNA biotinylated at the 5’ end. Oligonucleotides containing 24 bases and biotinylated at the 5’ end were obtained from Biotez (Berlin, Germany). The sequence of the oligonucleotides was as follows; 5’ Biotin-TAT CTG TTC ACC GCA AAT CTG TGG 3’. Alexa Fluor-488 FluoroNanogold 75 Streptavidin (SFNG) was obtained from Nanoprobes (New York, USA). The attached gold nanoparticles are 1.4 nm in diameter and contain approximately 80 Au atoms which are covalently attached to a streptavidin protein and Alexa Fluor-488 fluorophore.15 The manufacturer states that there is an average of one nanogold 80 particle to one streptavidin protein. Although streptavidin contains four biotin binding sites, only one or two of these sites may be accessible to the biotin in this case due to the steric restraints created by the presence of the nanogold and fluorophore. 15  The labelling of the biotinylated DNA was achieved by adding 85 an excess of SFNG to the biotinylated DNA in a polypropylene vial. The reaction mixture was prepared by adding SFNG (1.33 µM, 83 µl) to the 5’ biotinylated DNA solution (2.24 µM, 26 µl). Thus the reaction mixture contained: 58.7 pmoles biotinylated DNA and 110 pmoles streptavidin which equated to 442 pmoles of 90 biotin binding sites (assuming a total of 4 biotin binding sites per streptavidin molecule) and 17.12 mg/l gold. This reaction mixture therefore contained a potential 7.5 fold molar excess of biotin binding sites. The mixture was stored for approximately 48 hours at 40C.     95  Following the 48 hour reaction period, the reaction mixture was diluted so that the final solution contained 5.55 µg/l gold. This  Department of Chemistry, Loughborough University, Loughborough, Leicestershire, LE11 3TU Fax: 01509 223925; Tel: +44 (0) 1509 222572; E-mail: b.l.sharp@lboro.ac.ukCREATED USING THE RSC COMMUNICATION TEMPLATE (VER. 2.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS  2  |  [journal], [year], [vol], 00–00 This journal is © The Royal Society of Chemistry [year] diluted sample was then injected onto a HPLC column in order to separate the bound and unbound SFNG (see Table 1).  A C18 column was chosen to provide a rapid separation based on 100 size exclusion coupled with a small degree of hydrophobic interaction. A HP 1090 HPLC system (Agilent Technologies, Waldbronn, Germany) was employed; the column outlet was connected directly to the ICP nebuliser via a short length of 150 µM internal diameter Teflon tubing. The eluent flow was split post 105 column with a T-piece so that ~300 µl/min of the mobile phase entered the nebuliser. The remainder of the mobile phase was sent to waste. An Element 2XR high resolution ICP-MS instrument (Thermo Finnigan, Bremen, Germany) was employed for the determination of 197Au. The operating parameters are summarised 110 in Table 1.  A SFNG standard containing 3.66 µg/l Au was prepared from the stock SFNG solution. This standard solution was used for quantification and to establish the retention time of the unbound SFNG. Figure 1 shows the 197Au chromatogram of the SFNG 115 standard, which had a retention time of 1.57 minutes. The void volume of the column was determined with uracil, which had a retention time of 3.87 minutes.  This indicates that the SFNG was totally excluded from the pore space in the column. A very small peak was observed in the SFNG standard chromatogram at 1.06 120 minutes, which was attributed to carry over between samples. Gold has the disadvantage of being ‘sticky’ in nature and thus it can be difficult to completely remove all traces of the element between sample injections.  Figure 1 also shows the 197Au chromatogram of the DNA-SFNG 125 reaction mixture which contains two peaks. The first peak at 1.06 minutes corresponds to the DNA-SFNG conjugate and the unbound SFNG eluted at 1.60 minutes, which is consistent with the retention time of the unbound SFNG standard in Figure 1. The DNA-SFNG conjugate was the first species to elute because it is larger than the 130 unbound SFNG and therefore, likely to be more excluded from the C18 phase. More importantly, the bound SFNG carried a highly charged oligonucleotide, resulting in less hydrophobic interaction with the surface of the non-polar stationary phase. The peaks were integrated and the areas quantified using the SFNG standard. The 135 Au concentration in the peaks at 1.06 minutes and 1.60 minutes was 2.97 µg/l and 3.11 µg/l respectively, thus total Au concentration was calculated to be 6.08 µg/l in the injected sample. However, the actual Au concentration in the injected sample was 5.55 µg/l, suggesting that 109.5% of the Au was recovered from the 140 chromatographic phase. Errors in background correction were thought to be the reason behind the high recovery (<100%),  but this can be improved by improving the separation method. A control sample was also prepared which involved adding non-biotinylated oligonucleotide to SFNG. The reaction conditions 145 were similar to those described above. The control sample gave one peak with the retention time consistent with that of the unbound SFNG, as shown in Figure 1, thus implying that the reaction was site specific.  The SFNG was assayed to determine the number of Au atoms 150 per nanogold particle. Two Au calibration curves were prepared, one for SFNG and one for an elemental Au standard. The slopes of the calibration curves were 4510624 (x axis expressed as nM streptavidin) and 265217 (x axis expressed as ng/ml) for SFNG and elemental gold respectively. From these data it was calculated that 155 there were 86 Au atoms per nanogold particle. This figure is slightly higher than that given by the manufacturer. However, the manufacturer states that there is an average of one nanogold particle to each streptavidin molecule, more than one nanogold particle per protein would increase the average number of Au 160 atoms per particle and there may also be a small fraction of nanogold not bound to streptavidin. If either of these species were present they should be resolved from the DNA-SFNG conjugate during HPLC separation. No evidence of additional peaks was observed in any of the chromatograms. The figure of 86 Au atoms 165 per nanogold particle was used for further calculations, although this assay was only applied to one batch of SFNG, it is unknown whether the figure varies between batches.  From the chromatogram, it can be concluded that 53.5% of the nanogold and hence streptavidin was bound to DNA. Therefore, the 170 number of moles of bound streptavidin in the reaction mixture was 58.9 pmoles. If one biotinylated DNA molecule bound to one streptavidin protein, then 58.9 pmoles of DNA also bound. Considering 58.7 pmoles of DNA was in the reaction mixture, 100.3% of the DNA was labelled with SFNG. This data indicates 175 that with a 7.5 molar excess of potential binding sites, or a 7.5/4 ~2 fold molar excess of nanogold probe, only one binding site per protein molecule was occupied. This is the optimum labelling stoichiometry since it equates to one Au nano-particle (86 Au atoms) per oligonucleotide. If more than one oligonucleotide bound 180 to SFNG, more than one conjugate peak would have been  expected in the chromatogram since each additional oligonucleotide added to the SFNG would have resulted in an increase of 8KDa to the Table 1 Instrument Parameters    Fig. 1 197Au chromatogram of the SFNG standard containing 3.66 µg/l Au (top) and the DNA-SFNG reaction mixture containing 5.55 µg/l Au (bottom). Eluted from a Waters C18 µBondapak column, 300 mm x 3.9 mm with 5% Methanol isocratic elution, 10 µl injection volume. CREATED USING THE RSC COMMUNICATION TEMPLATE (VER. 2.1) - SEE WWW.RSC.ORG/ELECTRONICFILES FOR DETAILS  This journal is © The Royal Society of Chemistry [journal], [year], [vol], 00–00  |  3 conjugate mass. As the separation appeared to have some size exclusion character, such conjugates should have been resolved.  185  The Au background was consistent at approximately 12000 counts per second (cps), equivalent to ~ 70 ng/l of Au. This continuous flux of Au was thought to be hydrophobically bound SFNG from previous injections slowly eluting from the column. Repeated injections of nanogold produced an accumulation of Au 190 at the head of the column that could be reduced, but not totally removed by back flushing. The observed background Au signal may also have been enhanced due the presence of 5% methanol in the mobile phase which was shown to increase Au signals by ~16%. It has been suggested by Larsen et al.,16 that the presence of 195 carbon facilitates the ionisation of certain analytes in the plasma.16 This effect is well known for some elements with high ionisation potential such as Se and As.16 Rodushkin et al.,17 have also reported that the addition of methane to the spray chamber increased the signal intensity of some elements including Au.17 In addition, the 200 presence of the organic solvent reduces the surface tension of the Au solution, which may improve nebulisation efficiency.16    The chromatograms shown in Figure 1 exhibit a dip in the baseline at approximately 1.4 minutes. This reduction in signal was consistent and observed in all chromatograms, suggesting it was an 205 injection related event. The mobile phase consisted of 5% methanol but the samples were diluted in deionised water. From the order of elution in Figure 1, it can be established that the DNA-SFNG conjugate eluted first followed by the sample solvent, which contained a higher proportion of water compared to the mobile 210 phase, and finally the unbound SFNG eluted. If the background Au was hydrophobically bound then the elution of the water solvent (a weaker eluent than methanol) would transiently reduce the Au flux from the column. Further, reduced carbon load and higher surface tension of the water would reduce the Au signal further causing the 215 temporary reduction in baseline signal.   The final aspect considered was the enhancement in signal due to SFNG labelling compared to that obtained by direct monitoring of 31P. To calculate the enhancement factor, two calibration curves were prepared; one for 197Au and one for 31P. The slopes of the 220 calibration equations were 265765 and 1082 for 197Au and 31P respectively with the concentration axes expressed in µg/l. By comparing the gradients from the two sets of data, it was established that Au gave a 246 times greater response compared to P. The sensitivity of ICP-MS depends upon the number of similar 225 isotopes in the sample, so the number of  P and Au atoms also has to be considered. Each DNA-SFNG conjugate contained 24 P atoms from the DNA phosphate backbone and 86 Au atoms. Hence, each DNA-SFNG conjugate will have had 3.6 times more Au atoms compared to P. Therefore, an 882 times increase in signal 230 was observed for the Au labelled oligonucleotides. As indicated above, the detection limit for 31P is typically 50 ng/l, or given that P accounts for ~ 10% by mass of DNA, 500 ng/l for DNA. The amplification achieved here lowers that to ~ 500 pg/l or an estimated 5 fg for a 10 μl injection. The amplification factor  235 improves for smaller nucleic acids, but deteriorates for larger nucleic acids in proportion to the number of P atoms.  The enhancement in signal requires that the nanogold particles are completely ionised in the plasma and thus behave as Au atoms. Once the average number of Au atoms per nanogold particle was 240 established, Au calibration curves for SFNG and elemental Au standards were compared. The gradients for the two sets of calibration data were 266226 and 265217 (x axis expressed as µg/l in both cases) for SFNG and elemental Au respectively. The calibration data confirmed that SFNG gave a similar response to 245 the atomic Au solutions and is therefore ionised efficiently in the plasma. Recent work has suggested that for the plasma to process particles in a truly composition independent fashion requires them to be less than 90 nm in size (based upon the analysis of glass)18 which is much greater than the 1.4 nm in diameter of the nano-250 particles.   It has been demonstrated that nucleic acids can be labelled with Au nanoparticles and subsequently analysed by ICP-MS. Nanoparticle labelling coupled with elemental MS has several advantages. The problems of polyatomic interferences that are 255 encountered with 31P detection are avoided. The bio-molecule signal is greatly enhanced by the higher sensitivity for 197Au compared with 31P and the presence of ~86 gold atoms per oligonucleotide. The data shown in this communication also demonstrates that, unlike other techniques, ICP-MS can be utilised 260 to establish mass balance for the nano-particle label thereby ensuring that all the labelled species from the reaction are both detected and quantified. A further benefit of the elemental analysis approach is that, post-separation, the stability of the molecular species is not important since only the label is detected and at this 265 stage quantification can be undertaken using simple acidic Au standards.8 The data presented here show that ICP-MS is an ideal complement to organic MS for the study of nucleic acids and offers enhanced detection and quantification. References 270 1 D. Pröfrock, P. Leonhard and A. Prange, J. Anal. At. Spectrom., 2003, 18, 708-713. 2 M. Wind and W. D. Lehmann, J. Anal. At. Spectrom., 2004, 19, 20-25. 3 W. Brüchert and J. Bettmer, J. Anal. At. Spectrom., 2006, 21, 1271-275 1276. 4 N. Jakubowski, R. Lobinski and L. Moens, J. Anal. At. Spectrom., 2004, 19, 1-4. 5 R. Lobinski, D. Schaumlöffel and J. Szpunar, Mass Spectrom. Rev., 2006, 25, 255-289. 280 6 D. Pröfrock, P. Leonhard, S. Wilbur and A. Prange, J. Anal. At. Spectrom., 2004, 19, 623-631. 7 D. R. Bandura, V. I. Baranov and S. D. Tanner, Anal. Chem., 2002, 74, 1497-1502. 8 V. I. Baranov, Z. A. Quinn, D. R. Bandura and S. D. Tanner, J. Anal. 285 At. Spectrom., 2002, 17, 1148-1152. 9 P. D. Winship, PhD. Doctoral thesis, Loughborough University, Department of Chemistry, 2006 10 V. I. Baranov, Z. A. Quinn, D. R. Bandura and S. D. Tanner, Anal. Chem., 2002, 74, 1629-1636. 290 11 J. Bettmer, N. Jakubowski and A. Prange, Anal. Bioanal. Chem., 2006, 386, 7-11. 12 E. Morag, E. A Bayer and M. Wilchek, Biochem. J. 1996, 316, 193-199. 13 A. Merkoci, M. Aldavert, G. Tarrasón, R. Eritja and S. Alegret, Anal. 295 Chem., 2005, 77, 6500-6503. 14 C. W. Dieffenbach, T. M. J. Lowe and G. S. Dveksler, PCR Methods Applic., 1993, 3, 30-37. 15 Nanoprobes Incorporated, 20/06/07, http://www.nanoprobes.com 16 E. H. Larson, J. Anal. At. Spectrom., 1994, 9, 1099-1105. 300 17 I. Rodushkin, P. Nordlund, E. Engström and D. C. Baxter, J. Anal. At. Spectrom., 2005, 20, 1250-1255. 18 H. R. Kuhn, M. Guillong and D. Günther, Anal. Bioanal. 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Nano-particle labelling of nucleic acids for enhanced detection by inductively-coupled plasma mass spectrometry (ICP-MS)

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Nano-particle labelling of nucleic acids for enhanced detection by inductively-coupled plasma mass spectrometry (ICP-MS)

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