Multifunctional Nanoparticles for Biomedical Applications

IMIKRID an Integrated Microfluidic Diagnostic System

  • Multifunctional Nanoparticles for Biomedical ApplicationsMultifunctional Nanoparticles for Biomedical Applications
  • Multifunctional Nanoparticles for Biomedical Applications
  • Fig.1: Principle of multifunctional SiO2-based nanoparticles which enable the conjugation of marker-specific antibodies or relevant antigens and are tailored to both the sensor and the microfluidics of the diagnostic system.
  • Fig. 2: ζ-potential of non-modified and functionalized SiO2-based nanoparticles as a function of pH.
  • Fig.3: (A) Oriented conjugation of recombinant antibody single chain Ki4 SNAP Tag fusion via benzylguanine-modified NP and (B) confocal microscopy with luminescent SiO2-NPs coupled to Ki4-SNAP on L540 cells. Antibody-modified NPs in the cell membrane glow green; the cell nucleus stained with Draq5 (red). Overlay confirms that NPs specifically bind to the tumor-specific markers integrated in the cell membrane as a result of an antibody-antigen reaction.

Today, the prospects of surviving cancer or cardiovascular diseases are relatively high if the disease is diagnosed as early as possible. Improved early detection methods based on molecular markers could help patients receiving personalized therapies earlier than it is the case presently. Multifunctional nanoparticles enable the conjugation of marker-specific antibodies or relevant antigens owing to their size which is comparable to that of biomolecules and to their extraordinary properties.

Introduction
Cardiovascular and tumor diseases are the most frequent causes of death in Europe. One focus is on the prevention of these diseases. But a more precise and reliable diagnosis at an earlier stage could also save lives. For such an early diagnosis - long before the first macroscopically visible symptoms appear - a suitable test method, based on serological markers, is required.
While it is already possible to detect disease-specific biomarkers (substances which are produced by the body and indicate the presence and development of a disease) in blood serum, current technologies fail to offer adequate levels of detection sensitivity and accuracy, especially in the initial stages of a disease, when the concentrations of the respective substances in the patients' body fluids are extremely low. They can also not reflect that the "alarm-triggering" concentrations may vary from one patient to another. In addition, they are generally complex, time-consuming and cost-intensive. Valuable time is lost before patients can be treated on the basis of their individual symptoms, and often there is nothing more to be done than just to monitor the progression of the disease.
We focused our work on multifunctional particle systems which are able to bind biomarkers or encapsulate active substances or drugs and will open up new pathways in individualized diagnoses and therapies. The great potential of nanoparticles (NPs) as a diagnostic tool was recently demonstrated by the joint research project IMIKRID (Integrated microfluidic diagnostic systems), which was funded by the BMBF (Federal Ministry of Education and Research). The aim of the research project was to create a technology platform for the development of an integrated microfluidic diagnostic system.

Alongside other detection methods, the objective was to integrate a significantly more sensitive form of antibody-based biomarker analysis in a diagnostic chip, thus enabling the creation of extremely compact, portable and modular diagnostic systems capable of supplying clinically relevant measurement results within a matter of minutes.

All-round Concept for Early Diagnosis
One of the goals of IMIKRID was to develop a portable multi-sensor system capable of simultaneously detecting relevant tumor-specific and cardiological markers with a detection sensitivity beyond the current limit of 10-11 mol/l. The core component of the overall system is a microfluidic chip equipped with micro-channels through which a blood sample is circulated. The goal is to employ antibody-antigen reactions to measure even the smallest concentration of disease-specific markers in the blood serum in vitro, and to achieve a 100-fold improvement over the detection accuracy offered by current state-of-the-art methods.
The important part of the diagnostic chip are multifunctional SiO2-based NPs that are specifically adapted to the sensors and the microfluidic channels, with a spacer being used to position the NPs in the middle of the sample fluidic flow in order to increase the sensitivity of the diagnostic system. NPs act as a binding partner for the antibodies/antigens and help to amplify the signal in a fluid environment (fig. 1). The reaction of the particle-bound antibodies with the antigen results in a change in the charge on the particle surface, which is registered by the sensor chip via the NP.

Multifunctional Nanoparticles
To this end, we have developed multifunctional dye-doped SiO2-based NPs that enable the conjugation of specific biomolecules such as antibodies. The key factors here are the size, shape and surface of the NPs, since these are the geometric parameters that determine the type and quantity of antigens that can subsequently be captured and the level of precision that can be achieved when measuring the marker concentration in a microfluidic environment. Obtaining proof of the successful binding of an antibody during development of the NPs requires a visual check. For this purpose, the NPs were labeled with a luminescent dye. Synthesis of the SiO2 particles, which are between 60 and 200 nanometers in size, is conducted by wet chemical methods using sol-gel technology [1, 2]. Dye molecules are incorporated homogeneously into the silica matrix by covalent attachment. As a consequence, NPs show significant resistance to photobleaching and dye leakage. NPs doping with luminescent substances also opens up the option of using an optical sensor system.
The resulting NPs are subsequently modified with various chemical functionalities such as amine and carboxyl functions using conventional functionalization methods [3 - 5]. The presence of functionalities is analyzed qualitatively and quantitatively by a variety of characterization methods, including ζ-potential or dye-test (fig. 2) [6, 7]. These reactive groups facilitate the targeted coupling of capture biomolecules such as antibodies to the particle surface. The functionality of the bound antibodies was verified by subsequent biological tests. To this end, surface modified luminescent NPs are coupled to full-length antibodies as well as recombinant antibody formats (single chain fragments of variable regions: scFv).

Nanoparticle Based Immuno Detection Assay
To test the versatility of the coupling procedure, we used carboxyrhodamine labeled SiO2-NPs. CD30 receptor was chosen as tumor-associated target antigen highly overexpressed on the surface of Hodgkin lymphoma cells. The recombinant extracellular part of human CD30L as well as scFv Ki4, a high affinity antibody fragment generated from a monoclonal anti-CD30 antibody, has been shown to target L540 Hodgkin lymphoma cells efficiently [9, 10]. The anti-CD30 scFv Ki4 was coupled to these NPs by SNAP-Tag technology which allows equipping a given biological molecule with many different dyes, with biotin or with other substrates [8]. The Tag is derived from the human DNA-repair enzyme O(6)-alkylguanine DNA alkyltransferase. It rapidly, specifically, and covalently binds substrates that contain O(6)-benzylguanine. The technology has been applied in a variety of experimental systems, ranging from in-cell labeling of tagged proteins to the immobilization of proteins on chip surfaces. The labeling and binding properties of antibody conjugated NPs were analyzed by confocal microscopy. They showed specific attachment to the membrane of CD30+ L540 cells (fig. 3). No binding of NPs was observed on CD30- U937 cells (negative control).

Conclusion
Over the last year, individual modules of the integrated microfluidic diagnostic system have undergone successful testing on the basis of molecular markers that are present in low concentrations in patients‘ blood serum. The multifunctional SiO2-based NPs enable the conjugation of marker-specific antibodies or relevant antigens. This confirmed that targeted coupling of the antibody to the particle surface does indeed take place. It was demonstrated that the antibodies remain active and are able to bind various biomarker antigens as intended. The biofunctionalized NPs are tailored to both the sensor system and the microfluidics of the diagnostic system. This visual proof of the successful binding of an antibody during development of the NPs opens up the option of an optical sensor system.

Authors:
Dr. S. Dembski, Researcher, Fraunhofer ISC, Wuerzburg, Germany
Dr. C. Gellermann, Leader of Competence Team Nanoparticle and Composites, Fraunhofer ISC, Wuerzburg, Germany
Dr. J. Probst, Head of Business Unit Life Science, Fraunhofer ISC, Wuerzburg, Germany
Dr. T. Klockenbring, Researcher, Fraunhofer IME, Aachen, Germany
Prof. Dr. Dr. S. Barth, Head of Division Molecular Biology, Fraunhofer IME, Aachen, Germany

References:
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[2] Gellermann C. et al.: J. Sol-Gel Sci Technol 8, 173 (1997)
[3] Van Blaaderen A. et al.: J Colloid Interface Sci 156, 1 (1993)
[4] Waddell T. G. et al.: J Am Chem Soc 103, 5303 (1981)
[5] Zhao X. et al.: Proc Nat Acad Sci U.S.A 101, 15027 (2004)
[6] Schiestel T. et al.: J Nanosc Nanotechnol 4, 504 (2004)
[7] Udenfriend S. et al.: Science 178, 871 (1972)
[8] Kampmeier F. et al.: Bioconjugate Chem 20, 1010, (2009)
[9] Huhn M. et al.: Cancer Res 61, 8737 (2001)
[10] Klimka A. et al.: Br. J. Cancer 80, 1214 (1999)

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Contact

Fraunhofer-Institut für Silicatforschung ISC
Neunerplatz 2
97082 Würzburg
Germany

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