Karolien Jans

Specific Cell Targeting with Nanobody Conjugated Branched Gold Nanoparticles for Photothermal Therapy

Branched gold nanoparticles are potential photothermal therapy agents because of their large absorption cross section in the near-infrared window. Upon laser irradiation they produce enough heat to destroy tumor cells. In this work, branched gold nanoparticles are biofunctionalized with nanobodies, the smallest fully functional antigen-binding fragments evolved from the variable domain, the VHH, of a camel heavy chain-only antibody. These nanobodies bind to the HER2 antigen which is highly expressed on breast and ovarian cancer cells. Flow cytometric analysis and dark field images of HER2 positive SKOV3 cells incubated with anti-HER2 conjugated branched gold nanoparticles show specific cell targeting. Laser irradiation studies reveal that HER2 positive SKOV3 cells exposed to the anti-HER2 targeted branched gold nanoparticles are destroyed after five minutes of laser treatment at 38 W/cm(2) using a 690 nm continuous wave laser. Starting from a nanoparticle optical density of 4, cell death is observed, whereas the control samples, nanoparticles with anti-PSA nanobodies, nanoparticles only, and laser only, do not show any cell death. These results suggest that this new type of bioconjugated branched gold nanoparticles are effective antigen-targeted photothermal therapeutic agents for cancer treatment.

Fiber optic SPR biosensing of DNA hybridization and DNA–protein interactions

In this paper we present a fiber optic surface plasmon resonance (SPR) sensor as a reusable, cost-effective and label free biosensor for measuring DNA hybridization and DNA-protein interactions. This is the first paper that combines the concept of a fiber-based SPR system with DNA aptamer bioreceptors. The fibers were sputtered with a 50nm gold layer which was then covered with a protein repulsive self-assembled monolayer of mixed polyethylene glycol (PEG). Streptavidin was attached to the PEGs carboxyl groups to serve as a versatile binding element for biotinylated ssDNA. The ssDNA coated SPR fibers were first evaluated as a nucleic acid biosensor through a DNA-DNA hybridization assay for a random 37-mer ssDNA. This single stranded DNA showed a 15 nucleotides overlap with the receptor ssDNA on the SPR fiber. A linear calibration curve was observed in 0.5-5 microM range. A negative control test did not reveal any significant non-specific binding, and the biosensor was easily regenerated. In a second assay the fiber optic SPR biosensor was functionalized with ssDNA aptamers against human immunoglobulin E. Limits of detection (2nM) and quantification (6nM) in the low nanomolar range were observed. The presented biosensor was not only useful for DNA and protein quantification purposes, but also to reveal the binding kinetics occurring at the sensor surface. The dissociation constant between aptamer and hIgE was equal to 30.9+/-2.9nM. The observed kinetics fully comply with most data from the literature and were also confirmed by own control measurements.

Stability of mixed PEO-thiol SAMs for biosensing applications.

The secret of a successful affinity biosensor partially hides in the chemical interface layer between the transducer system and the biological receptor molecules. Over the past decade, several methodologies for the construction of such interface layers have been developed on the basis of the deposition of self-assembled monolayers (SAMs) of alkanethiols on gold. Moreover, mixed SAMs of polyethylene oxide (PEO) containing thiols have been applied for the immobilization of biological receptors. Despite the intense research in the field of thiol SAMs, relatively little is known about their biosensing properties in correlation with their long-term stability. Especially the impact of the storage conditions on their biosensing characteristics has not been reported before to our knowledge. To address these issues, we prepared mixed PEO SAMs and tested their stability and biosensing performance in several storage conditions, i.e., air, N2, ethanol, phosphate buffer, and H2O. The quality of the SAMs was monitored as a function of time using various characterization techniques such as cyclic voltammetry, contact angle, grazing angle Fourier-transform infrared spectroscopy, and X-ray photoelectron spectroscopy. In addition, the impact of the different storage conditions on the biosensor properties was investigated using surface plasmon resonance. Via the latter technique, the receptor immobilization, the analyte recognition, and the nonspecific binding were extensively studied using the prostate specific antigen as a model system. Our experiments showed that very small structural differences in the SAM can have a great impact in their final biosensing properties. In addition it was shown that the mixed SAMs stored in air or N2 are very stable and retain their biosensor properties for at least 30 days, while ethanol appeared to be the worst storage medium due to partial oxidation of the thiol headgroup. In conclusion, care must be taken to avoid SAM degradation during storage to retain typical SAM characteristics, which is very important for their general use in many proposed applications.

Poly(acrylic acid)-stabilized colloidal gold nanoparticles: synthesis and properties

Combining the intriguing optical properties of gold nanoparticles with the inherent physical and dynamic properties of polymers can give rise to interesting hybrid nanomaterials. In this study, we report the synthesis of poly(acrylic acid) (PAA)-capped gold nanoparticles. The polyelectrolyte-wrapped gold nanoparticles were fully characterized and studied via a combination of techniques, i.e. UV-vis and infrared spectroscopy, dark field optical microscopy, SEM imaging, dynamic light scattering and zeta potential measurements. Although PAA-capped nanoparticles have been previously reported, this study revealed some interesting aspects of the colloidal stability and morphological change of the polymer coating on the nanoparticle surface in an electrolytic environment, at various pH values and at different temperatures.

Shape-controlled synthesis of NIR absorbing branched gold nanoparticles and morphology stabilization with alkanethiols.

Gold nanoparticles are ideal candidates for clinical applications if their plasmon absorption band is situated in the near infrared region (NIR) of the electromagnetic spectrum. Various parameters, including the nanoparticle shape, strongly influence the position of this absorption band. The aim of this study is to produce stabilized NIR absorbing branched gold nanoparticles with potential for biomedical applications. Hereto, the synthesis procedure for branched gold nanoparticles is optimized varying the different synthesis parameters. By subsequent electroless gold plating the plasmon absorption band is shifted to 747.2 nm. The intrinsic unstable nature of the nanoparticles morphology can be clearly observed by a spectral shift and limits their use in real applications. However, in this article we show how the stabilization of the branched structure can be successfully achieved by exchanging the initial capping agent for different alkanethiols and disulfides. Furthermore, when using alkanethiols/disulfides with poly(ethylene oxide) units incorporated, an increased stability of the gold nanoparticles is achieved in high salt concentrations up to 1 M and in a cell culture medium. These achievements open a plethora of opportunities for these stabilized branched gold nanoparticles in nanomedicine.

Label-free genosensor based on immobilized DNA hairpins on gold surface

In this report, we demonstrate a label-free genosensor based on DNA hairpins coupled to gold coated sensor surfaces. The hairpin probes were labeled with a thiolated moiety for immobilization at the 5 end and with a fluorophore for signal transduction at the 3 end. In the absence of the complement, the fluorophore is quenched by energy transfer to the gold surface. Addition of the target sequence leads to the hairpin unfolding, and releases the fluorescent signal. This built-in property, using a gold film as both the immobilizing substrate and quenching agent, has the advantage of simplicity in design and ease of further integration. Our results showed that lengths of both the stem and the loop structures have significant effects on the sensor performance. Hybridization kinetics was investigated for various probe/target lengths and concentrations. An optimized hairpin probe gave a fluorescent signal increase of 39 folds after hybridization, which is much higher than the earlier reported results. A limit of detection (LOD) down to 0.3 nM for the complementary target DNA detection has been achieved. The developed sensor was further successfully applied for the detection of single-base mismatch targets, as well as for the direct detection of PCR products.

Increased stability of mercapto alkane functionalized Au nanoparticles towards DNA sensing

The use of gold nanoparticles (GNPs) in bioassays is often hampered by their colloidal stability. In this study, gold nanoparticles coated with different mercapto alkanes were investigated towards their stability. Hereto, the effects of the alkane chain length (5-11 methylene groups), the type of functional end-group (-OH or -COOH) and the amount of incorporated poly-ethylene oxide units (none, 3 or 6) on the GNP stabilization was evaluated. Based on these results, an optimal mercapto alkane (HS(CH(2))(11)PEO(6)COOH) was selected to increase the colloidal stability up to 2 M NaCl. Furthermore, it was proved that this mercapto alkane is ideally suited to enhance the stability of DNA functionalized GNPs in high electrolytic hybridization buffers. The effectiveness of these DNA functionalized GNPs was demonstrated in a sandwich assay using a surface plasmon resonance biosensor. The superior stability of these nanoparticles during hybridization may lead to enhanced biosensor technologies.

Synthesis of PEGylated Magnetic Nanoparticles With Different Core Sizes

Tailoring the properties of superparamagnetic nanoparticles (MNPs) is essential for various nano-based biological applications. Having control over the properties of the MNPs permits a maximum flexibility. Starting from monodisperse iron oxide MNPs produced by thermal decomposition, we report on the optimization and characterization of a first and second seed mediated growth step by varying the surfactant amount and by optimizing the heating steps. We demonstrate the ability to gradually increase the size of crystalline MNPs from 6 over 9 to 12 nm with an improving monodispersity as demonstrated by Transmission Electron Microscopy, Dynamic Light Scattering and X-ray diffraction. The magnetic properties of the MNPs, studied by Vibrating Sample Magnetometry, were in concert with their size increase. We also show the functionalization of these particles with polyethylene glycolated silanes, to render the MNPs stable in water. Different characterization techniques, namely Transmission Electron Microscopy, Dynamic Light Scattering, Fourier-transform InfraRed, Thermo gravimetric analysis and X-ray Photoelectron Spectroscopy, confirmed the successful engraftment of the silanes on the MNPs surface. In conclusion, the proposed route of step-wise synthesis in combination with silane functionalization allows fine tuning the physical properties of iron oxide MNPs for applications in an aqueous environment.

A simple double-bead sandwich assay for protein detection in serum using UV–vis spectroscopy

In this study a double-bead sandwich assay, employing magnetic nanoparticles and gold nanoparticles is proposed. The magnetic nanoparticles allow specific capturing of the analyte in biological samples, while the optical properties of the gold nanoparticles provide the signal transduction. We demonstrated that a major improvement in the assay sensitivity was obtained by selecting an optimal gold nanoparticle size (60 nm). A detection limit of 5-8 ng/mL, a sensitivity of 0.6-0.8 (pg/mL)(-1) and a dynamic range of 3 orders of magnitude were achieved without any further amplification using the detection of prostate specific antigen in serum as a model system. The proposed assay has the ability to be easily implemented within a microfluidic device for point-of-care applications whereby the readout can be executed by a fast and cheap optical measurement.

Impact of pre-concentration to covalently biofunctionalize suspended nanoparticles

The effective biofunctionalization of nanoparticles is crucial for biomedical applications. In this study we investigated the covalent biofunctionalization of magnetic nanoparticles based on carbodiimide activation. An important aspect in the covalent biofunctionalization of nanoparticles has been neglected, namely pre-concentration. Exploiting the electrostatic attraction forces between a protein and the nanoparticle surface will favor the covalent immobilization. We showed that low ionic strength buffers with a pH slightly lower than the pI of the selected biomolecules is needed to increase the yield of covalent immobilization. Additionally, it is demonstrated that the covalently immobilized proteins are bioactive, relying on a sandwich assay using gold nanoparticles as reporter labels.