The Gizeli Group

Acoustic waves for biosensing and innovation in healthcare

Research

By on 24th November 2016

Overview

Our work is highly multidisciplinary in nature, lying at the interface of molecular biology, biophysics, micro-engineering and nano-biotechnology. We are using acoustic waves as the means to study biological molecules and their interactions. We are also employing acoustic wave devices for the development of integrated platforms for molecular diagnostics. Our research is driven by scientific curiosity and innovation. The former in order to elucidate basic biological issues such as the structure of biomolecules and its significance in biological processes or the mechanism of cells’ interaction with solid substrates; the latter in order to provide useful clinical and diagnostic tools for improving people’s healthcare. Currently, we are actively engaged in the technology transfer of our novel findings to commercial products through the creation of a spin-off company dedicated to biosensors and point-of-care diagnostics.

Biophysical Studies

Funding agents: Horizon2020, HFSP, FP7

A major focus of our research is the elucidation of the way by which acoustic waves interact with biological molecules attached to the device surface and specifically, the molecular mechanisms involved in the interaction processes leading to acoustic energy dissipation. In our analysis, we consider that biomolecules attached to the device surface via an anchor are subject to a surface perturbation as a result of the wave propagation in the underlying substrate. We, therefore, imagine individual biomolecules being forced to oscillation which in turn produces a drag force between the moving biomolecule and the surrounding liquid.

 

Study of the conformation of surface-attached DNA molecules

We developed a novel theory which showed that acoustic measurements can be directly related to the intrinsic viscosity of a biomolecule attached to the device surface; the latter, in turn, is a hydrodynamic property that depends strongly on the molecule’s shape and size or, in other words, conformation. The theory has been tested by resolving via acoustic measurements the conformation of pre-designed double stranded DNA molecules of the same shape (rod) but different size and also molecules of the same size (90 bp) but of various conformations (double stranded “straight” and “curved”, triplex “straight”) (Biophys J 2008, Biosens Bioelectron 2008). The conformation of other structures such as the open or closed “cross” shape of a Holliday Junction DNA (Nano Lett 2010), or “spaghetti” type single-stranded DNA (Anal Chem 2012) have also been studied. Excellent results are also obtained in the case of the more complex triple-helical DNA with pre-designed “straight” and “bent” conformations. For the first time, acoustic sensors have been turned from mere mass detectors to quantitative structure measuring tools. The theory was tested experimentally providing a general proof of the hydrodynamic nature of acoustic wave/biomolecule interaction, for both DNA (Anal Chem 2016) and proteins (Chem Comm 2016).

Figure 1

DNA hybridization

The hydrodynamic nature of acoustic wave sensing and its subsequent sensitivity to molecular conformation was demonstrated to be well-suited for hybridization studies. Changes in the conformation of single stranded DNA molecules during their hybridization by a complementary strand can be easily recorded acoustically; from such measurements one can study the kinetics of the interaction and, more importantly, detect mismatches between the two strands (Anal Chem 2012). The detection of multiple miRNA targets with a single probe, based on differences in the conformation of the hybridized products, was also demonstrated in an assay suitable for multiplexing (Chem Comm 2015).

Cellular Studies

Funding agents: FP7, GSRT, Ministry of Education

Despite the widespread use of biosensors with soluble analytes, limited data exist on the detection of whole cells and cell-bound membrane receptor interactions. Acoustic wave biosensors have an important advantage over optical biosensors as they can offer analysis of cell-surface interactions at the molecular level (Cell Mol Life Sci 2012). Due to the confinement of the wave close to the interface (~100 nm), the sensor can focus on the protein-protein binding that mediates cell-substrate interactions; the bulk cell mass does not affect the acoustic signal so that non-specific adsorption of cells can be distinguished from specific interactions of interest. The observed sensitivity of acoustic damping to the number of cell/surface specific bonds provides a unique sensing mechanism for investigating membrane interactions (Biophys J 2008, Biosens Bioelectron 2010). Moreover, the acoustic signal change, together with a modified 3D kinetic analysis, can be used to measure detailed kinetics and derive both two-dimensional association and dissociation rate constants in a fast and simple way.

Cell adhesion of malignant cells on surfaces

Cell adhesion of malignant cells on surfaces

Cancer is a wide spread devastating condition where tools for high precision determination of the malign phenotype of cells and metastatic potential is crucial for treatment. Cell adhesion properties are of particular interest for cancer diagnosis since they are able to reflect the progressive state of cell-matrix and cell-cell loosening interactions. We are investigating differences in cell adhesion during the interaction of benign and malignant cells on inorganic surfaces and protein-modified Au; typical proteins used to cover the surface include collagen, fibrinogen and fibronectin. Our aim is to follow acoustically cell adhesion and correlate physicochemical parameters with the degree of cancer differentiation. Recently we demonstrated the ability to screen between normal and cancer thyroid cells through comparative adhesion studies on two different surfaces (Sensing Bio-Sensing Research 2016). This work was performed in collaboration with Dr E. Anastasiadou from the BRFAA, Greece.

Bacteria detection on modified surfaces

Over the past decade there has been an immense effort to develop new bioassays and biosensors for the rapid detection of food- and water-borne pathogens which remain a major cause of disease and mortality throughout the world. Within this framework, antibodies are used as the means to achieve bacterial capturing to the sensor substrate. Current work in our group focuses on the development of optimized acoustic device surfaces for bacteria detection by employing different types of protocols using monoclonal/polyclonal antibodies or aptamers, prior to the addition of the bacteria-containing sample (Salmonella Typhimurium and mutant strains). In collaboration with Dr E. Gogolides group from the NCSR-Demokritos, plasma micro- and nano-textured surfaces have been developed and used as alternative means for enhancing antibody binding, leading potentially to higher bacteria attachment on corrugated immuno-surfaces (Sens Actuat B: Chemical 2016). This work is also carried out in collaboration with Dr B. Dupuy from the Pasteur Institute in France.

Nano-biotechnology

Funding agents: : GSRT, FP7

Acoustic waves have a typical wavelength of few tenths of micrometers. The study of the intrinsic properties of nano-entities attached to the device surface is carried out in our lab using acoustic wave devices operating at several frequencies, i.e., from 5 to 300 MHz. In order to have spatial control of the immobilized molecules, the latter are bound through a single anchor (normally a PEG, thiol or DNA) to specific attachment points separated from each other by a certain distance. Through this single point attachment, biomolecules are presented to the surface in a suspended way as discrete particles, allowing the preservation of their native conformation. The above methodology is used for the immobilization of proteins and DNA molecules and subsequent study of changes of their conformation under well-controlled conditions.

 

DNA nanoswitches

Molecular nano-switches have been developed and studied as part of our nano-biotechnology studies. One such example is the DNA Holliday-junction; structure transitions between an “open” and “closed” state (mediated by magnesium) have been measured acoustically and the actual shape was deduced by independent intrinsic viscosity measurements (Nano Lett 2010). It has to be noticed that such a change is hard to be observed with label-free methods or even microscopy given the very small size (~ 10 nm) of the molecule.

Structural changes of intrinsically disordered proteins

Acoustic wave devices’ sensitivity to molecular hydrodynamic properties can be applied to the study of conformational changes of proteins such as the intrinsically disordered E.coli membrane protein ZipA. By attaching the protein to a supported lipid bilayer through a His-tag anchor, we showed that the protein was sensitive to the solution’s ionic strength, through the corresponding stretching or contraction of the unstructured domain (Chem Comm 2016). This conformational change was reflected on the change of the acoustic signals as they also followed the ionic-strength induced change of ZipA conformation, with an impressive sensitivity of 1.8 nm or less.

Devices and integrated platforms

Funding agents: Horizon2020, FP7, GSRT

Biosensors group has extensive know-how and experience in laboratory research with acoustic wave sensors (Surface Acoustic Wave (SAW) and QCM). The Love wave biosensor, a SAW-based waveguide device originally developed by Prof. E. Gizeli (Sensor Actuat 1992, Patent WO9201931), is a system routinely used for the propagation and detection of shear acoustic waves in a frequency range of 100 to 300 MHz. Today, the lab is also using a plethora of different commercially available or home-made devices to test novel concepts on acoustic sensing and develop integrated platforms. An ongoing research interest involves the design and manufacturing of acoustic wave devices both in a single and multi-channel array format. Devices in an array format are desirable since they require low volumes of consumables, thus, creating the potential for multiple testing. Acoustic SAW-type biochips are currently developed in collaboration with Dr JM. Friedt from Femto-ST Institute and the Franche-Comté University, France and our industrial partner Senseor, France.

Novel device geometries

In addition to the Love wave devices, a new project involves the design and fabrication of Lamb acoustic wave biosensors. The goal of this work is the fabrication of novel acoustic chips using materials such as Si, GaN and AlN (Appl Phys Lett 2010); this choice of materials will make the sensors compatible with compound semiconductor processing techniques, in contrast to the standard piezoelectric (quartz and lithium niobate etc) materials applied so far. This means that the sensor may serve as an element to a larger system (i.e., a Lab-on-a-Chip). This approach, developed in collaboration with Dr G. Konstantinidis from the Microelectronics Research Group, IESL-FORTH, Greece offers the advantage of providing a fully monolithically integrated system easily applicable to biodetection and point-of-care diagnostics.

Microfluidics-on-SAW and integrated diagnostic platforms

The future of biomedicine is strongly associated with the development of sensitive detection tools that take advantage of current advancements in engineering. The field of biomedical engineering builds upon mathematics, physics and biochemistry in order to provide solutions to medicine and the biomedical sciences. Among the research interests of our lab is the development of highly integrated and user-friendly systems that are based on a lab-on-a-chip concept and are capable of generating faster and more sensitive results. We have revolutionized acoustic sample detection by introducing the microfluidic-on-SAW (μF-on-SAW) concept; instead of carrying out the traditional “one-sample-per-sensor” detection strategy, we designed and developed a special microfluidic module, which comprises a parallel multi-channel configuration (J MEMS 2008). The advantage of this approach, developed in collaboration with Dr A. Tserepi from NCSR-Demokritos, is that, in a standardized and cost-effective way, the number of microchannels on each SAW device can vary from 4 to 10 according to the desired application, reaching the “many-samples-per-sensor” regime. The potential of the μF-on-SAW platform has been demonstrated during the fast, sensitive and reproducible detection of four cardiac markers (Anal Chim Acta 2011). Combination of the μF-on-SAW with Dip Pen nanolithography also resulted in the fast and efficient pre-functionalization of the microfluidic areas (Analyst 2012). The development of efficient microfluidic modules on SAW is an ongoing effort in our lab, currently carried out in collaboration with our industrial collaborator Jobst Technologies, Germany.

Molecular diagnostics for point-of-care

Funding agents: Horizon2020, FP7, GSRT

Research and medical laboratories normally rely on solid phase hybridization assays and real-time PCR to answer biological problems regarding gene expression profiling, determination of viral load in clinical samples, DNA and RNA quantification, bacterial identification, SNP genotyping and pharmaco-genomics. Both techniques are based either on non-specific or sequence-specific fluorescent reporters that generate a signal reflecting on the amount of the PCR product; detection and quantification of fluorescently labeled targets require expensive instrumentation and sophisticated algorithms in some cases. In our lab, we have developed a new approach for genetic testing which retains the advantages of PCR and overcomes its detection disadvantages thanks to the application of a novel acoustic approach for amplicon identification and quantification. Our approach relies on the detection of DNA conformation rather than mass, in a label-free manner. It also takes advantage of the direct detection of dsDNA amplicons rather than the hybridization of a ss-target to a surface-immobilized probe, thus, eliminating problems related to the optimization of probe immobilization and density as well as the need for temperature control. A considerable advantage of the method is the ability to achieve multiplexing of two or more targets by modulating their length (Sci Rep 2013).

The implementation of our acoustic methodology in real samples is a strong indicator that the method can be readily applied in routine DNA analysis. Its potential has been demonstrated in three different cases:

  1. Detection of an insecticide-resistant mutation in the ace-1 gene of Anopheles gambiae, the major malaria vector (Sci Rep 2013)
  2. Quantification of the change in the expression levels of the ABCA1 gene in the liver of mice which was induced by a synthetic ligand (Sci Rep 2013)
  3. Detection of 4 different mutations in BRCA1 and BRCA2 genes that could serve as a focused genetic screening test for breast cancer (Anal Meth 2013)

Our intention is to combine for the first time the detection power of our acoustic methodology with the advantages of on-chip polymerase chain reaction to develop an integrated platform for label-free DNA diagnostics. In the on-chip PCR, a reaction mixture is loaded on a microchip and is driven through different microchannels, which are constantly held at three different temperatures. This feature combined with the reduced thermal capacity of the chip leads to rapid thermal equilibrium of the PCR mixture allowing for fast thermocycling with low power consumption.

In comparison to the hours required to run a PCR with most bench-scale thermocyclers due to their high thermal mass and low heating and cooling rates, the chip-based microscale PCR can perform the job within minutes. The ultimate goal of this work is to develop a fully integrated and autonomous system for DNA analysis, where DNA amplification and detection is performed on a single platform and in a fast and label free manner. Such a system is currently under development for the detection of foodborne pathogen bacteria in milk and other samples in a total time of less than 4 hrs (as opposed to the 1-3 days normally required with current techniques). This ambitious work, funded by the EC in two follow up projects (Love Food and LoveFood2Market) is carried out in collaboration with several partners in academia (Dr A.Tserepi and Dr E.Gogolides from NCSR-Demokritos, Dr B. Depuy from Pasteur Inst. and Dr Z. Bilkova from Pardubice Un.) and industry (Jobst Technologies and Senseor).

Our ultimate goal is to develop integrated biosensing platforms that could be used directly in real human, food or environmental samples, such as blood, urine, milk, meat, soil etc. To achieve this, we are working towards the development of biocompatible surfaces that could selectively and with high sensitivity detect the amplified DNA in a complex medium. Such an example is a surface coated with the co-polymer PEG-polylysine and is used for the selective binding of amplified DNA in the presence of lysed cells, proteins and the PCR amplification medium (i.e., primers, enzymes and Triton).

Our strong interest and commitment towards the technology transfer of acoustic biosystems to clinical diagnostics is also illustrated in our involvement in the development of a platform for the detection of circulating DNAs and their mutations in blood samples. The LiqBiopSens project, also funded by the EC through a Horizon2020-Innovation program, promises the delivery of a fast, direct, real time and inexpensive instrument that has the potential to save thousands of lives through the early and accurate colorectal cancer detection.