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Multivalent linkers for improved covalent binding of oligonucleotides to dye-doped silica nanoparticles
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Nanotechnology 26 365703 (http://iopscience.iop.org/0957-4484/26/36/365703) View the table of contents for this issue, or go to the journal homepage for more
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Nanotechnology Nanotechnology 26 (2015) 365703 (12pp)
doi:10.1088/0957-4484/26/36/365703
Multivalent linkers for improved covalent binding of oligonucleotides to dye-doped silica nanoparticles S M Kelleher1, R I Nooney1, S P Flynn1, E Clancy1,2, M Burke2, S Daly1, T J Smith1,2, S Daniels1,3 and C McDonagh1,4 1
Biomedical Diagnostics Institute, Dublin City University, Glasnevin, Dublin 9, Ireland National Centre for Biomedical Engineering Science, National University of Ireland Galway, University Road, Galway, Co. Galway, Ireland 3 National Centre for Plasma Science and Technology, Glasnevin, Dublin 9, Ireland 4 School of Physics, Dublin City University, Glasnevin, Dublin 9, Ireland 2
E-mail: [email protected]. Received 23 April 2015, revised 1 July 2015 Accepted for publication 23 July 2015 Published 21 August 2015 Abstract
This paper describes the fabrication of oligonucleotide-coated Cy5-doped silica nanoparticles using a combination of multivalent linkers and their use in surface-based DNA sandwich hybridization assays. Dipodal silane is introduced as a means to fabricate amine-coated silica nanoparticles and its advantages compared to monopodal silanes are discussed. The use of dipodal silane in conjunction with three different polymer linkers (oxidized dextran, linear and 8-arm polyethylene glycol (PEG)) to immobilize single-stranded DNA to Cy5-doped nanoparticles is investigated and dynamic light scattering measurements and Fourier transform infrared spectroscopy are used to follow the progression of the functionalization of the nanoparticles. We observe a significant improvement in the binding stability of the singlestranded DNA when the dipodal silane and 8-arm PEG are used in combination, when compared to alternative conjugation strategies. Both 8mer and 22mer oligonucleotides are securely conjugated to the high-brightness nanoparticles and their availability to hybridize with a complementary strand is confirmed using solution-based DNA hybridization experiments. In addition, a full surface-based sandwich assay demonstrates the potential these nanoparticles have in the detection of less than 500 femtomolar of a DNA analogue of micro RNA, miR-451. S Online supplementary data available from stacks.iop.org/NANO/26/365703/mmedia Keywords: bioconjugation, dipodal silanes, high brightness silica nanoparticles, 8-arm polyethylene glycol, hybridization assays, miR451 (Some figures may appear in colour only in the online journal) wide range of fluorescent dyes can be loaded inside silica NPs for high sensitivity detection of biomarkers or multiplexing applications using either the reverse micro-emulsion or Stöber method [7–11]. For point-of-care (POC) devices, dye-doped silica NPs are attracting significant attention and have already been integrated into commercial products [12–15]. With a large amount of dye encapsulation, the increased brightness of these NPs results in improved signal-to-noise ratios compared
Introduction Highly fluorescent dye-doped silica nanoparticles (NPs) have great potential as labels in biomedical diagnostics such as for the detection of nucleic acid or protein biomarkers. Silica NPs are optically transparent and can be modified using a wide range of organosilane linkers for bioconjugation [1–3]. In addition, silica NPs are biocompatible [4, 5], photostable [6] and protect fluorescent dyes from molecular quenchers. A 0957-4484/15/365703+12$33.00
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© 2015 IOP Publishing Ltd Printed in the UK
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Figure 1. Schematic showing the conjugation approach for nanoparticle functionalization using activation silanes, ABisTES and APTES, and linkers, linear PEG, 8-arm PEG and oxidized dextran.
to free dye labels [8]. Furthermore, synthesis can be achieved using inexpensive alkoxide precursors at room temperature [16]. When developing POC assays, the binding of the active biomolecule (which interacts directly with the target) to the surface of the high-brightness nanoparticle is also favourable as it limits the number of steps required to run the full assay. Another important factor that must be taken into account is that the biomolecules e.g. oligonucleotides, antibodies and enzymes, are bound in a stable manner to the nanoparticle surface; for this, covalent immobilization is preferable to physisorption [17]. However, it is vital that with such immobilization the biomolecules not only remain attached under vigorous washing in aqueous conditions, but that they also remain active and available for binding. When choosing immobilization strategies for linking of biomolecules to silica nanoparticles, the use of silanes for surface activation is commonplace. In particular, aminopropyltriethoxysilane (APTES) linkers are used to provide the surface of the silica with amine groups, to which biomolecules can be directly linked. However, there is evidence that these APTES groups can become chemically unstable at high and low pH, and at raised temperatures, conditions often experienced during assay development [18–20]. In this work, we investigated the use of the dipodal silane, bis [3-(triethoxysilyl) propyl] amine, (ABisTES), which increases crosslinking density of silanes on silica surfaces i.e. ABisTES
is able to form six bonds to the NP per silane compared to three for APTES (see figure 1) [21]. The principal of improving the stability of binding of linkers by increasing the number of attachment points has been previously demonstrated with multidentate thiol ligands for attaching oligonucleotides to gold nanoparticles [22–25]. Dipodal silanes participate in covalent binding with silica surfaces and have previously been recommended in the fields of catalysis and separation science when such surfaces are used under aqueous conditions, where the hydrolytic stability of the siloxane bond between the surface and functionalization group is unstable [19, 20]. We hypothesize that the use of ABisTES to provide the surface of the silica nanoparticle with amine functionalisation would improve the chemical stability of these groups on the surface, by limiting the rate at which hydrolysis occurred. We are not aware of previous work on the use of dipodal silanes to functionalize silica nanoparticles. The binding of biomolecules directly to surfaces can have a negative effect on the availability of the biomolecules to interact with target molecules due to restricted movement and steric hinderance. In these instances, the use of spacers and linkers provide may not only improved binding stability but also improve the availablity of bound biomolecules for interaction with the required targets. In addition, selection of the correct linkers can provide nanoparticles with increased colloidal stability [17, 26, 27]. Multivalent linkers have 2
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shown to be highly effective for stable binding of biomolecules to surfaces: in previous work using multivalent dendrimer polymer linkers for application in immunoassay, significant improvements in antibody binding and assay performance were observed [28]. Multivalent polymer linkers have also improved binding and availability of oligomers on surfaces [29–32]. We are currently developing nucleic-acid conjugated, dye-doped silica NPs for use in POC diagnostic systems for the detection of micro RNA biomarkers. When working with silica NPs where APTES was used to provide the amine surface and small, linear linkers to immobilize oligonucleotide probes, we observed substantial loss of the oligonucleotides from the surface after washing (see figure S1 in supplementary information). This led us to investigate the method of using dipodal silanes combined with multivalent linkers multi-arm polyethylene glycol (8PEG) and oxidized dextran (OxDex) to immobilize the oligonucleotides. Linear polyethylene glycol (LPEG) was investigated as a linear comparison. Figure 1 outlines the stages at which the different silanes and linkers were employed. These linkers were chosen to provide improved availability of the bound oligonucleotides, as well as possible reduction in non-specific binding (NSB), a factor crucial to control when developing surfacebased assays [33, 34]. 8PEG has previously been used in the formation of polymer networks for tissue engineering and in nanoaggregate synthesis but it has not been utilized as a functional coating for NPs until now [35–37]. OxDex has been used previously as a coating for lab-on-a-chip devices to immobilize antibodies and for the immobilization of streptavidin to silica NPs, and dextran has been used previously to coat NPs for calcium sensing applications [38–40]. The key features of this paper include (i) the novel use of a dipodal silane to improve functionality of silica nanoparticles, (ii) use of 8-arm PEG linker for greater oligonucleotide surface loading and (iii) use of these nanoparticles to detect of a DNA analogue of the micro RNA, miR-451 at femtomolar concentrations in a surface-based hybridization assay.
0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4, at 25°C), disodium phosphate (>99%), monosodium phosphate (>99%), hydrochloric acid (1.0 N), 2-(N-morpholino)ethanesulfonic acid hydrate (MES hydrate, >99.5%), ammonium hydroxide solution (28% w/w in water), Nhydroxysuccinimide (NHS) (98%), O,O′-bis[2-(N-succinimidyl-succinylamino)ethyl] polyethylene glycol (LPEG-NHS) (2000 Da), sodium periodinate (ACS reagent, 99.8%), diglycolic anhydride (90%), potassium bromide ( 99%), phosphoric acid (ACS reagent, 85 wt% in H2O), glutaraldehyde (50% wt in water), sodium borohydride (98%), Micro90, and albumin from bovine serum (BSA, freeze dried powder, 96%) were all purchased from Sigma Aldrich, Dublin, Ireland. Deionized water (20 mV) up to 168 h in solution; demonstrating that the amine groups were still present on both samples and that hydrolysis was not occurring on these samples under these conditions at neutral pH. However, upon purification and resuspension in phosphoric acid solution, ABisTES-coated NPs were colloidally stable whereas the APTES-coated NPs were observed to aggregate in less than two minutes (see figure S4 in supplementary information). A similar improvement in colloidal stability was also observed when bidentate thiol ligands were attached to gold nanoparticles; however, the thiol-gold bond is reversible and the use of this bidentate ligand did not lead to improved bioconjugation [46]. The size of the ABisTEScoated NPs was determined after 5 and 30 min using DLS and was found to be 106±28.4 nm and 84.9±25.1 nm respectively. It was not possible to measure the size of the APTES-coated NPs due to rapid aggregation. In summary, the DLS analysis demonstrated that the amine functionalization for both the APTES and ABisTES NPs was stable in phosphoric acid and that significant hydrolysis of the conjugated silanes was not observed. Although the reason for investigating ABisTES as an alternative to APTES was primarily to
investigate hydrolytic stability, it was this significant improvement in colloidal stability that led us to recommend ABisTES linkers for surface activation as a first step in silica bioconjugation. Linker optimization
We proceeded to use ABisTES-coated nanoparticles in conjunction with multivalent linkers to attempt to improve the binding of the oligonucleotides. We selected OxDex and eight-arm polyethylene glycol (8PEG) as multivalent linkers, and LPEG as a monovalent comparison. After attachment to the NP as described in the materials and methods section, the OxDexNP, LPEGNP and 8PEGNP were then conjugated to amine-terminated, a Cy3-labelled oligonucleotide (Entries a, b or g in table 1, depending on the application). Figure 3 shows the Cy3 fluorescence signal from nanoparticles coated with 22mer ssDNACy3 (Entry a, table 1) bound via the three different linkers, initially and after three washes in SDS buffer. The rapid aggregation of the APTES particles described above could also be the reason for the poor conjugation of the DNA observed in our initial experiments as we did not observe to have the same difficulty in achieving secure binding using ABisTES. All three samples bound a large amount of DNACy3 (79 strands for LPEGNP, 81 strands for OxDexNP and 85 for 8PEGNP) and a strong fluorescence signal was seen. The first washing step in SDS buffer reduced the amount of ssDNA bound to the surface of each sample (66 strands for LPEGNP, 62 strands for OxDexNP and 83 for 8PEGNP). It was assumed that during this wash step most of the non-covalently bound ssDNA was removed. Further washes show that the DNA binding stabilizes after three washes, with 57 strands per NP calculated for the 8PEGNP indicating that it provided the strongest binding of all linkers. Combined with the ABisTES functionalization, 8PEGNP not only initially bound more 7
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Figure 4. Zeta potential of 8PEG-coated nanoparticles in different buffers as measured by DLS. Error bars represent the standard deviations of calculated from the DeltaNano software.
oligonucleotides than the OxDexNP or LPEGNP samples but also and more importantly, the NP was shown to retain the largest amount of ssDNA strands (approximately twice as much as OxDex or LPEG linked particles) after successive washing. We believe that the multivalency of the 8PEG linker allowed for multiple covalent bonds to be formed between the 8PEG and the amine coating on the NP as well as multiple ssDNA strands to bind to the 8PEG. This improved binding of the oligonucleotides agreed with our previous work on the use of multivalent linkers to attach antibodies to NP labels for use in immunoassays, where the use of multivalent linkers led to a higher efficiency in bioconjugation of detection antibodies [28, 47]. In the case of the LPEG linker, there was only one binding point available for attachment to the dipodal silane, which could have been more readily available for hydrolysis than binding points on a multivalent linker, resulting in fewer ssDNA strands be attached to the surface via covalent bonding. In the case of the OxDex linker, initial oligonucleotide binding was strong but levelled out after 2 washes to a value comparable to that of LPEG. We postulate that the decreased number of ssDNA conjugated to OxDex compared to the multivalent 8PEG was due to fewer functional groups available for binding, as approximately 40% of the alcohol groups on OxDex were converted to aldehydes during the 2 h oxidization reaction [45]. Some loss of functionality of the OxDex may also be attributed to the hydrolysis of the enamine group formed during the Schiff-base reaction in the SDS buffer. It was therefore concluded that the optimum conjugation combination was the dipodal functionalisation with the 8PEG linker (8PEGNP-DNA). A number of control experiments were carried out in order to ensure that the binding of the oligomers was covalent in nature. The zeta potential of 8PEGNP in different buffers (pH 7.0, or in the case of MES, pH 4.7) was calculated at room temperature and the results (figure 4) confirm that the nanoparticles are generally either neutral or negatively charged, making the physical adsorption of negatively charged oligonucleotides
highly unlikely. Further analysis of the stability of these silica nanoparticles can be found in other published work by this group [48]. Experiments were also performed without the addition of EDC to activate linkers during the conjugation steps and the binding of the oligonucleotide was not successful (see figure S5 in the supplementary information), demonstrating that the binding is indeed covalent and not merely physical. Finally, the robust binding of amine-terminated oligomers to the surface was demonstrated using a probe control where there was no terminal amine group. Figure S5 (supplementary information) also shows the lack of stable binding of this non-modified oligonucleotide to the 8PEGNP in the presence of EDC activation. This confirms that little or no physical adsorption of oligomers takes place on the surface of the 8PEGNP. In previous work by Delport et al, amine-functionalised ssDNA with an ATTO 647N flurophore label were coupled to silica NPs (∅=249 nm) using carbodiimide chemistry, in which 80 ssDNA oligomers were shown to attach per NP [49]. This corresponds to approximately one ssDNA strand per 2430 nm2 of silica surface. Using the ABisTES-8PEG system, we found that approximately 16 ssDNA are immobilized per unit area compared to 1 ssDNA strand using direct linking to a carboxyl functionalised surface (see materials and methods section for procedure). TEM images of the 8PEGNP are also shown in figure S3d (supplementary information) and the mean diameter of the particles was calculated to be 53±2 nm. Using carboxyl activated surface and direct linking, we attached four ssDNA strands per silica NP (∅=80 nm), corresponding to approximately 1 ssDNA strand per 5000 nm2 (data not shown); again this was significantly lower than that obtained using this multivalent approach. When calculating the number of DNA strands on the surface of the nanoparticle, we were careful to ensure that there was no quenching of signals from any Förster resonance energy transfer between the Cy3 on the surface and the Cy5 in the nanoparticle (see figure S6 in the supplementary information). 8
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Figure 5. (a) ssDNA targets and probes used to test hybridization. Upon hybridization of the complementary strand, dabcyl quencher will
reduce the fluorescence signal from the Cy3-labelled conjugated ssDNA; (b) fluorescent signal from Cy3-labelled 22mer and 8mer probes on ABisTES and APTES 8PEG NPs which have been hybridized with quencher probes. Graph shows the percentage DNA strands which were hybridized (fluorescent signal was quenched) and which were not (fluorescent signal remained unquenched).
manner to the NP, we attribute the large reduction in fluorescence to the dabcyl quencher being in very close proximity to the Cy3 dye. We believe that the target oligonucleotide is participating in full hybridization with both strands bound to the silica NP and that therefore, the ssDNA bound to the surface was available for hybridization for either direct binding or sandwich binding, and that steric interference or entrapment from the polymer coating did not affect the binding of the complementary strand. We also attribute the stability of the DNA strands on the APTES-coated particles to the use of the 8PEG linker. To exemplify the potential applicability of our assay, we applied it to the detection of a DNA analogue of the micro RNA, miR-451. miR-451 is widely dysregulated in human cancers and plays a critical role in tumourigenesis and tumour progression [50]. miR-451 is present in human plasma at picomolar concentrations [51]. miR-451 may therefore have potential is a non-invasive diagnostic and prognostic biomarker for cancer. A reporter probe (Entry g, table 1) was then bound to the NPs using the ABisTES-8PEG conjugation. These nanoparticles were then used in a surface-based sandwich assay using the complementary capture probe and target miR-451 analogue (Entries e and f, table 1). This sandwich assay (figure 6(a)) was developed using surface chemistry and immobilization methods which reduces any NSB of the nanoparticles. We observed that the 8PEG-coated nanoparticles provide the least amount of NSB to our surfaces when compared to other nanoparticles we used. The capture probe was immobilized, followed by the introduction of the target miR-451 DNA analogue. After this, either the reporter probe-coated Cy5 NPs or a Cy5-conjugated reporter probe (Entry h, table 1) (‘free dye’) was added. Any NSB of target, reporter probes or nanoparticles was minimized by adding BSA to the surface after capture probe immobilization,
Availability of conjugated DNA
It was important to ensure that the full oligomer chain bound to the NP was available for hybridization with its complementary strand and not sterically hindered by the hydrophilic, porous polymer coating. To demonstrate this, fluorescence quenching studies from hybridization with complementary DNA containing dabcyl quenchers were carried out. We performed these experiments on both ABisTES and APTES functionalized 8PEG-coated nanoparticles; this enabled us to directly compare the use of 8PEG with both silane linkers. Figure 5(a) shows a schematic of the two single-stranded reporter probes bound to the 8PEGcoated NP; one with a 22mer DNACy3 and another with an 8mer DNACy3 (Entries a and b, table 1). The complementary targets containing the dabcyl quencher were then added to this solution. For the 22-mer reporter probe, the target contained a dabcyl moiety at the 3′ end, and for the 8-mer reporter probe, an internal dabcyl was used (Entries c and d, table 1). After the complementary targets were introduced, the washing of the NPs (five times) ensured that any non-specifically bound target was removed. Figure 5(b) presents the hybridization availability as a percentage of probes covalently bound to each nanoparticle. For both the ABisTES and the APTES particles, the quenching effect can be seen by the considerable reduction in the signal from the dye-labelled oligonucleotides (88% loss of signal from the 22mer and 69% loss of signal from the 8mer for the ABisTES, and 79% loss of signal from the 22mer and 60% loss of signal from the 8mer for the APTES). The high negative charge of both the 8PEG-coated NPs and the oligonucleotide-coated NPs combined with a large amount of washing ensured that the target strand did not bind non-specifically. Knowing that the ssDNA is bound in a robust 9
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functionalisation improved colloidal stability over monofunctional APTES-coated particles and, in combination with multivalent 8-arm PEG linkers, significantly improved DNA conjugation stability. We have shown that oligonucleotides bound to these NPs were available for hybridization to direct binding or sandwich binding targets in solution phase with no NSB. Finally, a DNA sandwich hybridization assay was carried out using these DNA-conjugated high-brightness nanoparticles as the reporter and LOD of less than 500 fM, a greater than 7-fold increase in sensitivity than using the Cy5 dye alone. Acknowledgments This material is based upon works supported by Enterprise Ireland and Science Foundation Ireland under Grants 10/CE/ B1821, 14/TIDA/2334 and 14/TIDA/2369. Supplementary information Loss of DNA from APTES-coated nanoparticles. Cy3 free dye calibration curve. TEM images of functionalised nanoparticles. Aggregation of nanoparticles over time. Robustness of probe binding with no EDC/non-modified probes. FRET investigations.
Figure 6. (a) Schematic outlining the process of the sandwich assay;
(b) fluorescent signal measured for the sandwich assay using both reporter probe-coated Cy5-nanoparticles (‘nanoparticle’) and Cy5labelled reporter probes (‘free dye’). Error bars represent the standard deviations of three fluorescence intensity measurements. Equations used to calculate the limit of detection determined from the trendlines. Scanned at an instrument gain of 80.
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followed by deactivating any remaining glutaraldehyde groups with sodium borohydride prior to addition of target and reporter probes. The Perkin Elmer Scanner fluorescent signal from the final reading of the Cy5 reporter and Cy5 nanoparticles bound to the probe areas is presented in figure 6(b). The limit of detection (LOD) of the two methods was calculated using the equations presented on the graph, which were determined by the fit of the two trendlines and from the standard deviation of the fluorescent signal at a target concentration of 0 pM. The minimum signal from the 0 pM sample shows that there is no NSB of the nanoparticles to the probe area. The calculated LOD for the free dye assay was 3.58 pM of target, whereas the LOD of the nanoparticle assay was 478 fM of target miR-451 DNA analogue, indicating that the use of the Cy5 high-brightness nanoparticles results in greater than a 7-fold increase in sensitivity than using the Cy5 dye alone.
Conclusion Oligonucleotide-conjugated Cy5-doped silica nanoparticles were prepared which showed excellent efficiency for the binding of DNA. The use of novel dipodal ABisTES 10
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Multivalent linkers for improved covalent binding of oligonucleotides to dye-doped silica nanoparticles
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 Nanotechnology 26 365703 (http://iopscience.iop.org/0957-4484/26/36/365703) View the table of contents for this issue, or go to the journal homepage for more
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Nanotechnology Nanotechnology 26 (2015) 365703 (12pp)
doi:10.1088/0957-4484/26/36/365703
Multivalent linkers for improved covalent binding of oligonucleotides to dye-doped silica nanoparticles S M Kelleher1, R I Nooney1, S P Flynn1, E Clancy1,2, M Burke2, S Daly1, T J Smith1,2, S Daniels1,3 and C McDonagh1,4 1
Biomedical Diagnostics Institute, Dublin City University, Glasnevin, Dublin 9, Ireland National Centre for Biomedical Engineering Science, National University of Ireland Galway, University Road, Galway, Co. Galway, Ireland 3 National Centre for Plasma Science and Technology, Glasnevin, Dublin 9, Ireland 4 School of Physics, Dublin City University, Glasnevin, Dublin 9, Ireland 2
E-mail: [email protected]. Received 23 April 2015, revised 1 July 2015 Accepted for publication 23 July 2015 Published 21 August 2015 Abstract
This paper describes the fabrication of oligonucleotide-coated Cy5-doped silica nanoparticles using a combination of multivalent linkers and their use in surface-based DNA sandwich hybridization assays. Dipodal silane is introduced as a means to fabricate amine-coated silica nanoparticles and its advantages compared to monopodal silanes are discussed. The use of dipodal silane in conjunction with three different polymer linkers (oxidized dextran, linear and 8-arm polyethylene glycol (PEG)) to immobilize single-stranded DNA to Cy5-doped nanoparticles is investigated and dynamic light scattering measurements and Fourier transform infrared spectroscopy are used to follow the progression of the functionalization of the nanoparticles. We observe a significant improvement in the binding stability of the singlestranded DNA when the dipodal silane and 8-arm PEG are used in combination, when compared to alternative conjugation strategies. Both 8mer and 22mer oligonucleotides are securely conjugated to the high-brightness nanoparticles and their availability to hybridize with a complementary strand is confirmed using solution-based DNA hybridization experiments. In addition, a full surface-based sandwich assay demonstrates the potential these nanoparticles have in the detection of less than 500 femtomolar of a DNA analogue of micro RNA, miR-451. S Online supplementary data available from stacks.iop.org/NANO/26/365703/mmedia Keywords: bioconjugation, dipodal silanes, high brightness silica nanoparticles, 8-arm polyethylene glycol, hybridization assays, miR451 (Some figures may appear in colour only in the online journal) wide range of fluorescent dyes can be loaded inside silica NPs for high sensitivity detection of biomarkers or multiplexing applications using either the reverse micro-emulsion or Stöber method [7–11]. For point-of-care (POC) devices, dye-doped silica NPs are attracting significant attention and have already been integrated into commercial products [12–15]. With a large amount of dye encapsulation, the increased brightness of these NPs results in improved signal-to-noise ratios compared
Introduction Highly fluorescent dye-doped silica nanoparticles (NPs) have great potential as labels in biomedical diagnostics such as for the detection of nucleic acid or protein biomarkers. Silica NPs are optically transparent and can be modified using a wide range of organosilane linkers for bioconjugation [1–3]. In addition, silica NPs are biocompatible [4, 5], photostable [6] and protect fluorescent dyes from molecular quenchers. A 0957-4484/15/365703+12$33.00
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Figure 1. Schematic showing the conjugation approach for nanoparticle functionalization using activation silanes, ABisTES and APTES, and linkers, linear PEG, 8-arm PEG and oxidized dextran.
to free dye labels [8]. Furthermore, synthesis can be achieved using inexpensive alkoxide precursors at room temperature [16]. When developing POC assays, the binding of the active biomolecule (which interacts directly with the target) to the surface of the high-brightness nanoparticle is also favourable as it limits the number of steps required to run the full assay. Another important factor that must be taken into account is that the biomolecules e.g. oligonucleotides, antibodies and enzymes, are bound in a stable manner to the nanoparticle surface; for this, covalent immobilization is preferable to physisorption [17]. However, it is vital that with such immobilization the biomolecules not only remain attached under vigorous washing in aqueous conditions, but that they also remain active and available for binding. When choosing immobilization strategies for linking of biomolecules to silica nanoparticles, the use of silanes for surface activation is commonplace. In particular, aminopropyltriethoxysilane (APTES) linkers are used to provide the surface of the silica with amine groups, to which biomolecules can be directly linked. However, there is evidence that these APTES groups can become chemically unstable at high and low pH, and at raised temperatures, conditions often experienced during assay development [18–20]. In this work, we investigated the use of the dipodal silane, bis [3-(triethoxysilyl) propyl] amine, (ABisTES), which increases crosslinking density of silanes on silica surfaces i.e. ABisTES
is able to form six bonds to the NP per silane compared to three for APTES (see figure 1) [21]. The principal of improving the stability of binding of linkers by increasing the number of attachment points has been previously demonstrated with multidentate thiol ligands for attaching oligonucleotides to gold nanoparticles [22–25]. Dipodal silanes participate in covalent binding with silica surfaces and have previously been recommended in the fields of catalysis and separation science when such surfaces are used under aqueous conditions, where the hydrolytic stability of the siloxane bond between the surface and functionalization group is unstable [19, 20]. We hypothesize that the use of ABisTES to provide the surface of the silica nanoparticle with amine functionalisation would improve the chemical stability of these groups on the surface, by limiting the rate at which hydrolysis occurred. We are not aware of previous work on the use of dipodal silanes to functionalize silica nanoparticles. The binding of biomolecules directly to surfaces can have a negative effect on the availability of the biomolecules to interact with target molecules due to restricted movement and steric hinderance. In these instances, the use of spacers and linkers provide may not only improved binding stability but also improve the availablity of bound biomolecules for interaction with the required targets. In addition, selection of the correct linkers can provide nanoparticles with increased colloidal stability [17, 26, 27]. Multivalent linkers have 2
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shown to be highly effective for stable binding of biomolecules to surfaces: in previous work using multivalent dendrimer polymer linkers for application in immunoassay, significant improvements in antibody binding and assay performance were observed [28]. Multivalent polymer linkers have also improved binding and availability of oligomers on surfaces [29–32]. We are currently developing nucleic-acid conjugated, dye-doped silica NPs for use in POC diagnostic systems for the detection of micro RNA biomarkers. When working with silica NPs where APTES was used to provide the amine surface and small, linear linkers to immobilize oligonucleotide probes, we observed substantial loss of the oligonucleotides from the surface after washing (see figure S1 in supplementary information). This led us to investigate the method of using dipodal silanes combined with multivalent linkers multi-arm polyethylene glycol (8PEG) and oxidized dextran (OxDex) to immobilize the oligonucleotides. Linear polyethylene glycol (LPEG) was investigated as a linear comparison. Figure 1 outlines the stages at which the different silanes and linkers were employed. These linkers were chosen to provide improved availability of the bound oligonucleotides, as well as possible reduction in non-specific binding (NSB), a factor crucial to control when developing surfacebased assays [33, 34]. 8PEG has previously been used in the formation of polymer networks for tissue engineering and in nanoaggregate synthesis but it has not been utilized as a functional coating for NPs until now [35–37]. OxDex has been used previously as a coating for lab-on-a-chip devices to immobilize antibodies and for the immobilization of streptavidin to silica NPs, and dextran has been used previously to coat NPs for calcium sensing applications [38–40]. The key features of this paper include (i) the novel use of a dipodal silane to improve functionality of silica nanoparticles, (ii) use of 8-arm PEG linker for greater oligonucleotide surface loading and (iii) use of these nanoparticles to detect of a DNA analogue of the micro RNA, miR-451 at femtomolar concentrations in a surface-based hybridization assay.
0.0027 M potassium chloride and 0.137 M sodium chloride, pH 7.4, at 25°C), disodium phosphate (>99%), monosodium phosphate (>99%), hydrochloric acid (1.0 N), 2-(N-morpholino)ethanesulfonic acid hydrate (MES hydrate, >99.5%), ammonium hydroxide solution (28% w/w in water), Nhydroxysuccinimide (NHS) (98%), O,O′-bis[2-(N-succinimidyl-succinylamino)ethyl] polyethylene glycol (LPEG-NHS) (2000 Da), sodium periodinate (ACS reagent, 99.8%), diglycolic anhydride (90%), potassium bromide ( 99%), phosphoric acid (ACS reagent, 85 wt% in H2O), glutaraldehyde (50% wt in water), sodium borohydride (98%), Micro90, and albumin from bovine serum (BSA, freeze dried powder, 96%) were all purchased from Sigma Aldrich, Dublin, Ireland. Deionized water (20 mV) up to 168 h in solution; demonstrating that the amine groups were still present on both samples and that hydrolysis was not occurring on these samples under these conditions at neutral pH. However, upon purification and resuspension in phosphoric acid solution, ABisTES-coated NPs were colloidally stable whereas the APTES-coated NPs were observed to aggregate in less than two minutes (see figure S4 in supplementary information). A similar improvement in colloidal stability was also observed when bidentate thiol ligands were attached to gold nanoparticles; however, the thiol-gold bond is reversible and the use of this bidentate ligand did not lead to improved bioconjugation [46]. The size of the ABisTEScoated NPs was determined after 5 and 30 min using DLS and was found to be 106±28.4 nm and 84.9±25.1 nm respectively. It was not possible to measure the size of the APTES-coated NPs due to rapid aggregation. In summary, the DLS analysis demonstrated that the amine functionalization for both the APTES and ABisTES NPs was stable in phosphoric acid and that significant hydrolysis of the conjugated silanes was not observed. Although the reason for investigating ABisTES as an alternative to APTES was primarily to
investigate hydrolytic stability, it was this significant improvement in colloidal stability that led us to recommend ABisTES linkers for surface activation as a first step in silica bioconjugation. Linker optimization
We proceeded to use ABisTES-coated nanoparticles in conjunction with multivalent linkers to attempt to improve the binding of the oligonucleotides. We selected OxDex and eight-arm polyethylene glycol (8PEG) as multivalent linkers, and LPEG as a monovalent comparison. After attachment to the NP as described in the materials and methods section, the OxDexNP, LPEGNP and 8PEGNP were then conjugated to amine-terminated, a Cy3-labelled oligonucleotide (Entries a, b or g in table 1, depending on the application). Figure 3 shows the Cy3 fluorescence signal from nanoparticles coated with 22mer ssDNACy3 (Entry a, table 1) bound via the three different linkers, initially and after three washes in SDS buffer. The rapid aggregation of the APTES particles described above could also be the reason for the poor conjugation of the DNA observed in our initial experiments as we did not observe to have the same difficulty in achieving secure binding using ABisTES. All three samples bound a large amount of DNACy3 (79 strands for LPEGNP, 81 strands for OxDexNP and 85 for 8PEGNP) and a strong fluorescence signal was seen. The first washing step in SDS buffer reduced the amount of ssDNA bound to the surface of each sample (66 strands for LPEGNP, 62 strands for OxDexNP and 83 for 8PEGNP). It was assumed that during this wash step most of the non-covalently bound ssDNA was removed. Further washes show that the DNA binding stabilizes after three washes, with 57 strands per NP calculated for the 8PEGNP indicating that it provided the strongest binding of all linkers. Combined with the ABisTES functionalization, 8PEGNP not only initially bound more 7
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Figure 4. Zeta potential of 8PEG-coated nanoparticles in different buffers as measured by DLS. Error bars represent the standard deviations of calculated from the DeltaNano software.
oligonucleotides than the OxDexNP or LPEGNP samples but also and more importantly, the NP was shown to retain the largest amount of ssDNA strands (approximately twice as much as OxDex or LPEG linked particles) after successive washing. We believe that the multivalency of the 8PEG linker allowed for multiple covalent bonds to be formed between the 8PEG and the amine coating on the NP as well as multiple ssDNA strands to bind to the 8PEG. This improved binding of the oligonucleotides agreed with our previous work on the use of multivalent linkers to attach antibodies to NP labels for use in immunoassays, where the use of multivalent linkers led to a higher efficiency in bioconjugation of detection antibodies [28, 47]. In the case of the LPEG linker, there was only one binding point available for attachment to the dipodal silane, which could have been more readily available for hydrolysis than binding points on a multivalent linker, resulting in fewer ssDNA strands be attached to the surface via covalent bonding. In the case of the OxDex linker, initial oligonucleotide binding was strong but levelled out after 2 washes to a value comparable to that of LPEG. We postulate that the decreased number of ssDNA conjugated to OxDex compared to the multivalent 8PEG was due to fewer functional groups available for binding, as approximately 40% of the alcohol groups on OxDex were converted to aldehydes during the 2 h oxidization reaction [45]. Some loss of functionality of the OxDex may also be attributed to the hydrolysis of the enamine group formed during the Schiff-base reaction in the SDS buffer. It was therefore concluded that the optimum conjugation combination was the dipodal functionalisation with the 8PEG linker (8PEGNP-DNA). A number of control experiments were carried out in order to ensure that the binding of the oligomers was covalent in nature. The zeta potential of 8PEGNP in different buffers (pH 7.0, or in the case of MES, pH 4.7) was calculated at room temperature and the results (figure 4) confirm that the nanoparticles are generally either neutral or negatively charged, making the physical adsorption of negatively charged oligonucleotides
highly unlikely. Further analysis of the stability of these silica nanoparticles can be found in other published work by this group [48]. Experiments were also performed without the addition of EDC to activate linkers during the conjugation steps and the binding of the oligonucleotide was not successful (see figure S5 in the supplementary information), demonstrating that the binding is indeed covalent and not merely physical. Finally, the robust binding of amine-terminated oligomers to the surface was demonstrated using a probe control where there was no terminal amine group. Figure S5 (supplementary information) also shows the lack of stable binding of this non-modified oligonucleotide to the 8PEGNP in the presence of EDC activation. This confirms that little or no physical adsorption of oligomers takes place on the surface of the 8PEGNP. In previous work by Delport et al, amine-functionalised ssDNA with an ATTO 647N flurophore label were coupled to silica NPs (∅=249 nm) using carbodiimide chemistry, in which 80 ssDNA oligomers were shown to attach per NP [49]. This corresponds to approximately one ssDNA strand per 2430 nm2 of silica surface. Using the ABisTES-8PEG system, we found that approximately 16 ssDNA are immobilized per unit area compared to 1 ssDNA strand using direct linking to a carboxyl functionalised surface (see materials and methods section for procedure). TEM images of the 8PEGNP are also shown in figure S3d (supplementary information) and the mean diameter of the particles was calculated to be 53±2 nm. Using carboxyl activated surface and direct linking, we attached four ssDNA strands per silica NP (∅=80 nm), corresponding to approximately 1 ssDNA strand per 5000 nm2 (data not shown); again this was significantly lower than that obtained using this multivalent approach. When calculating the number of DNA strands on the surface of the nanoparticle, we were careful to ensure that there was no quenching of signals from any Förster resonance energy transfer between the Cy3 on the surface and the Cy5 in the nanoparticle (see figure S6 in the supplementary information). 8
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Figure 5. (a) ssDNA targets and probes used to test hybridization. Upon hybridization of the complementary strand, dabcyl quencher will
reduce the fluorescence signal from the Cy3-labelled conjugated ssDNA; (b) fluorescent signal from Cy3-labelled 22mer and 8mer probes on ABisTES and APTES 8PEG NPs which have been hybridized with quencher probes. Graph shows the percentage DNA strands which were hybridized (fluorescent signal was quenched) and which were not (fluorescent signal remained unquenched).
manner to the NP, we attribute the large reduction in fluorescence to the dabcyl quencher being in very close proximity to the Cy3 dye. We believe that the target oligonucleotide is participating in full hybridization with both strands bound to the silica NP and that therefore, the ssDNA bound to the surface was available for hybridization for either direct binding or sandwich binding, and that steric interference or entrapment from the polymer coating did not affect the binding of the complementary strand. We also attribute the stability of the DNA strands on the APTES-coated particles to the use of the 8PEG linker. To exemplify the potential applicability of our assay, we applied it to the detection of a DNA analogue of the micro RNA, miR-451. miR-451 is widely dysregulated in human cancers and plays a critical role in tumourigenesis and tumour progression [50]. miR-451 is present in human plasma at picomolar concentrations [51]. miR-451 may therefore have potential is a non-invasive diagnostic and prognostic biomarker for cancer. A reporter probe (Entry g, table 1) was then bound to the NPs using the ABisTES-8PEG conjugation. These nanoparticles were then used in a surface-based sandwich assay using the complementary capture probe and target miR-451 analogue (Entries e and f, table 1). This sandwich assay (figure 6(a)) was developed using surface chemistry and immobilization methods which reduces any NSB of the nanoparticles. We observed that the 8PEG-coated nanoparticles provide the least amount of NSB to our surfaces when compared to other nanoparticles we used. The capture probe was immobilized, followed by the introduction of the target miR-451 DNA analogue. After this, either the reporter probe-coated Cy5 NPs or a Cy5-conjugated reporter probe (Entry h, table 1) (‘free dye’) was added. Any NSB of target, reporter probes or nanoparticles was minimized by adding BSA to the surface after capture probe immobilization,
Availability of conjugated DNA
It was important to ensure that the full oligomer chain bound to the NP was available for hybridization with its complementary strand and not sterically hindered by the hydrophilic, porous polymer coating. To demonstrate this, fluorescence quenching studies from hybridization with complementary DNA containing dabcyl quenchers were carried out. We performed these experiments on both ABisTES and APTES functionalized 8PEG-coated nanoparticles; this enabled us to directly compare the use of 8PEG with both silane linkers. Figure 5(a) shows a schematic of the two single-stranded reporter probes bound to the 8PEGcoated NP; one with a 22mer DNACy3 and another with an 8mer DNACy3 (Entries a and b, table 1). The complementary targets containing the dabcyl quencher were then added to this solution. For the 22-mer reporter probe, the target contained a dabcyl moiety at the 3′ end, and for the 8-mer reporter probe, an internal dabcyl was used (Entries c and d, table 1). After the complementary targets were introduced, the washing of the NPs (five times) ensured that any non-specifically bound target was removed. Figure 5(b) presents the hybridization availability as a percentage of probes covalently bound to each nanoparticle. For both the ABisTES and the APTES particles, the quenching effect can be seen by the considerable reduction in the signal from the dye-labelled oligonucleotides (88% loss of signal from the 22mer and 69% loss of signal from the 8mer for the ABisTES, and 79% loss of signal from the 22mer and 60% loss of signal from the 8mer for the APTES). The high negative charge of both the 8PEG-coated NPs and the oligonucleotide-coated NPs combined with a large amount of washing ensured that the target strand did not bind non-specifically. Knowing that the ssDNA is bound in a robust 9
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functionalisation improved colloidal stability over monofunctional APTES-coated particles and, in combination with multivalent 8-arm PEG linkers, significantly improved DNA conjugation stability. We have shown that oligonucleotides bound to these NPs were available for hybridization to direct binding or sandwich binding targets in solution phase with no NSB. Finally, a DNA sandwich hybridization assay was carried out using these DNA-conjugated high-brightness nanoparticles as the reporter and LOD of less than 500 fM, a greater than 7-fold increase in sensitivity than using the Cy5 dye alone. Acknowledgments This material is based upon works supported by Enterprise Ireland and Science Foundation Ireland under Grants 10/CE/ B1821, 14/TIDA/2334 and 14/TIDA/2369. Supplementary information Loss of DNA from APTES-coated nanoparticles. Cy3 free dye calibration curve. TEM images of functionalised nanoparticles. Aggregation of nanoparticles over time. Robustness of probe binding with no EDC/non-modified probes. FRET investigations.
Figure 6. (a) Schematic outlining the process of the sandwich assay;
(b) fluorescent signal measured for the sandwich assay using both reporter probe-coated Cy5-nanoparticles (‘nanoparticle’) and Cy5labelled reporter probes (‘free dye’). Error bars represent the standard deviations of three fluorescence intensity measurements. Equations used to calculate the limit of detection determined from the trendlines. Scanned at an instrument gain of 80.
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followed by deactivating any remaining glutaraldehyde groups with sodium borohydride prior to addition of target and reporter probes. The Perkin Elmer Scanner fluorescent signal from the final reading of the Cy5 reporter and Cy5 nanoparticles bound to the probe areas is presented in figure 6(b). The limit of detection (LOD) of the two methods was calculated using the equations presented on the graph, which were determined by the fit of the two trendlines and from the standard deviation of the fluorescent signal at a target concentration of 0 pM. The minimum signal from the 0 pM sample shows that there is no NSB of the nanoparticles to the probe area. The calculated LOD for the free dye assay was 3.58 pM of target, whereas the LOD of the nanoparticle assay was 478 fM of target miR-451 DNA analogue, indicating that the use of the Cy5 high-brightness nanoparticles results in greater than a 7-fold increase in sensitivity than using the Cy5 dye alone.
Conclusion Oligonucleotide-conjugated Cy5-doped silica nanoparticles were prepared which showed excellent efficiency for the binding of DNA. The use of novel dipodal ABisTES 10
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