Comm. Appl. Biol. Sci, Ghent University, 72/1, 2007
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USE OF NANO-PARTICLES IN BIOSENSING
F. DELPORT, B. VERBRUGGEN, J. LAMMERTYN
Biosensor Group, Division Mechatronics, Biostatistics and Sensors, K.U.Leuven,
Willem de Croylaan 42, BE-3001 Heverlee, Belgium
INTRODUCTION
New developments in biofunctionalisation of nanomaterials are a driving
force for innovation in bionanotechnology applications. Nanoparticles (NP’s)
functionalised with a layer of biomolecules such as proteins and DNA, play a
central role in the field of life sciences for their diagnostic and therapeutic
properties. They act as miniaturised biosensors or drug delivery vehicles.
The properties of the bioconjugated nanomaterials depend both on the physical and material properties of the nanomaterial (nanorods, quantum dots,
branched nanoparticles …) both also on the biomolecules attached to its
surface (enzymes, antibodies, DNA, etc.) In all cases it is important to accurately characterize the properties of the bioconjugated nanomaterial.
To maximise the efficiency of the NP's, the conjugation chemistry and
coating have to be optimised for every material, shape or size of the NP and
every type of biomolecule. We present a robust set of techniques for the immobilisation of different bio-molecules on NP’s and stress some points of
attention specifically for the use in bio-assays e.g., purification or biosensing. Finally we will present NP's applied in DNA and antibody based biosensing, designed for lab-on-a-chip purposes.
MATERIALS AND METHODS
Nanoparticles and reagentia
Silica NP’s (300nm) and magnetic silica NP’s (1 µm), both carboxyl functionalised were purchased respectively from Micromod (Rostock-Warnemuende,
Germany) and Chemicell (Berlin, Germany). Amine, biotin, 6-FAM and Atto
647N functionalised 15-40 bp DNA was obtained from Eurogentec (Luik,
Belgium). EDC (1-Ethyl-3-(3-Dimethylaminopropyl) carbodiimide Hydrochloride) originates from Pierce Biotechnology (Rockford, USA). NHS (NHydroxysuccinimide), ethanolamine, PEG (polyethylene glycol) and all other
chemicals were purchased from Sigma-aldrich (Bornem, Belgium). The ELISA kit for peanut allergen Ara h1 was bought from R-Biopharm AG (Darmstadt, Germany). All immobilisation reactions were performed in a 100 mM
MES (2-(N-morpholino)ethanesulfonic acid) buffer with pH=5.
Immobilisation methods
For all protein bioconjugation experiments EDC chemistry was applied1. A
quantity of NP was suspended in 0.15 mg/ml EDC to activate the carboxyl
groups. Proteins were added (varying from 100 pM to 1 nM) to the NP’s after
10 min. when most of the EDC had reacted. This prevents protein crosslinking, but lowers the binding efficiency of the reaction. The protein-NP mixture
was shaken for 90min. Since in this protocol no specific orientation of the
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proteins is favoured, some active sites are sterically hindered resulting in
less protein activity compared to proteins in free solution.
To immobilise DNA on the NP’s two strategies were followed. In the first,
biotinylated DNA was immobilised on streptavidin functionalised NP’s. The
streptavidin was bound following to the abovementioned protocol for protein
immobilisation. In a second DNA immobilisation strategy amine labelled
DNA was directly coupled to the carboxyl functionalised NP through EDC
chemistry. Amine labelled DNA has only one binding site and thus a lower
binding efficiency, but is therefore incapable of crosslinking to itself enabling
a one step reaction. Furthermore, higher densities of conjugated DNA (relative to the first strategy) on the NP surface were realised. This is required to
maximise the biosensing efficiency. To further improve the efficiency the
activated carboxyl groups were stabilised with NHS before reaction with the
primary amines. The optimal concentrations were found to be 12.5 mg/ml
for both the EDC and NHS. An advantage of this direct DNA binding is the
possibility to construct a backcoating layer, e.g. polyethylene glycol (PEG)2.
Since the MES conjugation buffer promoted, next to specific binding, also
unspecific binding of biomolecules to the surface a special washing buffer
was required. This had a neutral pH, 0.3 M NaCl, 30 mM Na3citrate and 5
g/l SDS (sodium dodecyl sulphate). To prevent sticking of unwanted biomolecules an amino-PEG layer was applied to the NP’s after DNA immobilisation, to fill the space between the DNA molecules. An excess of ethanolamine (25 mM for 30 minutes) was added after DNA immobilisation or optional PEGylation to block the activated carboxyl groups on the surface of the
NP’s. Ethanolamine binds all activated groups left on the surface and changes a charged carboxyl surface into a hydrophilic hydroxyl surface, preventing
unspecific binding.
Quantification of DNA on NP’s
A spectrophotometer (SpectraMax M2e, Molecular Devices, Toronto, Canada)
was used to measure fluorescently tagged DNA or coloured solutions of the
ELISA in 96 well microtiterplates. The number of particles was kept constant
during all measurements, as it was pointed out that they have an impact on
the fluorescent signal.
RESULTS
Protein immobilisation on NP’s
To proof the protein binding method and check the activity of the bound
proteins, two separate experiments were conducted. First a colour reaction
using peroxidase coupled antibodies (Pase Ab’s) and, second, the binding of
biotin labelled fluorescent DNA to immobilised streptavidin.
Using the protein binding protocol, anti Ara h1 antibodies tagged with
peroxidase were immobilised on silica particles of 300 nm. After washing the
NP’s - to remove unbound Pase Ab’s - and using the colouring kit a strong
yellow colour was observed, proving that Pase Ab’s were immobilised on the
Comm. Appl. Biol. Sci, Ghent University, 72/1, 2007
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NP’s and that they partially retained their catalytic properties (Figure 1 a). In
a second experiment, NP’s were coated with streptavidin in order to bind
biotin labelled biomolecules. In this case, varying amounts of biotin labelled
fluorescent DNA were used to prove the non-destructive immobilisation of
the streptavidin. When using an excess of streptavidin coated NP’s, most of
the DNA was bound to the NP’s (figure 1 b).
Figure 1: a) Catalytic effect of Peroxidase-antibodies coated NP’s. Blank samples are uncoated NP’s. b) Biotin-DNA immobilisation on streptavidin coated
NP’s. Error bars indicate a standard deviation.
DNA immobilisation on NP’s
The EDC/NHS immobilisation reaction of DNA to 2.5 mg/ml NP’s continues
for 24h (figure 2 a). The higher the added concentration of DNA, the more
DNA was bound and this with high linearity in a wide range (figure 2 b).
Great care had to be committed to work with pure samples, as unspecific
binding can rise up to 80% in this stage, before PEGylation and ethanolamine treatment.
Figure 2: a) Binding kinetics of DNA to NP’s via direct binding, using 25 nM
DNA. b) Equilibrium measurements of DNA immobilisation
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Biosensor assays
Using the selected immobilisation methods, biosensing assays were developed with both proteins and DNA. First an existing ELISA kit was converted
to an ELISA on bead, in order to measure more complex samples. Secondly a
DNA sensing assay is presented, using DNA hybridisation, to detect DNA of a
specific sequence.
ELISA on bead
A proof of principle experiment is presented using the peanut allergen Ara
h1 and polyclonal antibodies3. The antibodies were immobilised on silica
particles and mixed with samples containing Ara h1. After capturing most
allergens with the antibodies, the rest of the sample was washed away. In a
second step an excess of polyclonal antibodies coupled to peroxidase (Pase
Ab) was added for binding to the Ara h1 allergens. After washing and following the colouring protocol of the ELISA kit, an increased absorption with a
peak at 450 nm was measured, directly depending on the Ara h1 concentration. Figure 3 clearly shows the catalytic properties of Pase Ab coated NP’s
and the excess of Pase Ab. Notice the absence of colour in the two washing
steps, showing the strength of the bond.
Figure 3: Spectra of coloured solutions. The highest signal is the excess of
unbound peroxidase antibodies, the NP’s with bound peroxidase antibodies
have a signal strength linearly related to the Ara h1 concentration. The
washing steps contain no peroxidase antibodies and thus give no colour
reaction.
DNA detection and quantification
By immobilising DNA on NP’s, both through streptavidin-biotin or direct
immobilisation, it is possible to capture complementary strands in a sample
through hybridisation. During the first phases of the development, the more
flexible method of streptavidin-biotin was used with fluorescent DNA. To
minimise unspecific binding, further experiment were conducted using the
direct binding of DNA followed by PEGylation and ethanolamine treatment.
Comm. Appl. Biol. Sci, Ghent University, 72/1, 2007
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The following samples were prepared by conjugating 50 nM DNA of which
7.5 nM was immobilised (figure 2 b). The detection of complementary hybridised DNA is only linear in a very small region compared to the 7.5 nM immobilised DNA. The first graph, where samples were measured after 24h, shows
a saturation of 100% hybridisation in excess of complementary DNA. When
monitoring the kinetics, a high initial hybridisation rate depending on added
concentration is visible (figure 4 b). This allows DNA to be detected, even in
very short assays. Unspecific testing also demonstrates the PEG efficiency,
while unspecific binding was always less than 0.5% and partial matching
DNA samples were clearly discriminated from full matches (Figure not
shown).
Figure 4: a) Equilibrium measurements after 24h of hybridisation. B) Kinetic
measurement of DNA hybridisation for two different concentrations of DNA.
CONCLUSIONS
In this paper a set of methods is described for protein and DNA immobilisation on NP’s, for a large range of applications. It was shown that the proteins
retained both catalytic activity and binding capacity upon binding. The catalytic capacity of the antibody coupled peroxide was used to perform a NP
based ELISA assay for allergen detection. The streptavidin-DNA functionalised NP’s were used as a test case for DNA hybridisation or aptamer sensing.
To bind more DNA to the surface and prevent the unspecific binding of
DNA, a direct immobilisation strategy was followed. Using this method, completed with PEGylation and an ethanolamine treatment, DNA hybridisation
was studied quantitatively and qualitatively. More complicated biosensing
assay’s, based on enzymes, antibodies, DNA and aptamers are the next step.
Varying the size and material of the NP’s a broad range of applications becomes available, in particular in microfluidic biosensors.
REFERENCES
1. Hermanson, G. T. Bioconjugate Techniques; 2008.
2. Wattendorf, U.; Merkle, H. P. Journal of Pharmaceutical Sciences 2008, 97 (11),
4655-46693.
3. Park, D. L.; Coates, S.; Brewer, V. A.; Garber, E. A. E.; Abouzied, M.; Johnson, K.;
Ritter, B.; McKenzie, D. Journal of Aoac International 2005, 88 (1), 156-160.
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