Interdigitated electrode DNA biosensor for detection of food-borne pathogens by impedance spectroscopy

The commercial application of biosensors has had a significant impact in a number of areas, particularly in the field of medical diagnostics. But in spite of the great number of publications, only a few systems are commercially available in food analysis. We propose an innovative approach to achieve safer food products by creating a user-oriented device for the detection of food-borne pathogens. Our aim is to perform quality control along the food-chain distribution to protect the consumer's health against contaminations; the measurement is based on the detection by impedance spectroscopy of DNA extracted from bacteria. Our work focuses on the conception of a DNA biosensor able to detect the presence of specific DNA sequences from Salmonella bacteria. Within the European project GoodFood, the choice was made to use dairy products and seafood as starting materials, from which cell growth and DNA amplification were performed by our partners, to finally be measured by impedance spectroscopy with the DNA biosensor. The major advantages of this approach are the portability, ease-of-use and rapidity of the system. The sensor fabrication was performed in several steps: At first, the design and the clean-room fabrication was optimized for platinum interdigitated structures with 5 µm spacing. Once the production of the metal electrodes was standardized, the immobilization of the probe DNA (capturing the target DNA) on the sensor surface was performed by silanization. The selectivity of the sensor towards the target DNA was evaluated by fluorescence microscopy using fluorescently-labelled DNA target sequences. The biochip, consisting of 4 interdigitated electrodes, was connected to an impedance analyzer and a first series of measurements was done without DNA, to characterize the sensor in high ionic strength solutions. A next series was performed with probe DNA on the sensor surface, with decreasing concentrations of synthetic target DNA to reach the nanomolar detection limit of the system. Finally, a series of measurements was carried out with DNA target sequences extracted from food samples spiked with salmonella, to apply the biosensor within a "real" biological DNA sample.

Application of CMOS image sensors in optical sectioning and polarization microscopy

Jelena Mitic

In this thesis new imaging approaches for optical microscopy are proposed and studied. They are based on the use of dynamic structured illumination in combination with a demodulation detection concept employing CMOS image detectors. Two particular implementations are suggested: real-time optical sectioning microscopy and whole field quantitative polarization microscopy. Optical sectioning, i.e depth resolved imaging, allows observation of thin sections in volumetric samples. In its classical configuration the wide-field microscope does not allow optical sectioning of the investigated objects. In this thesis the optical sectioning in a wide-field microscope is achieved by illuminating the sample with a continuously moving single spatial frequency pattern, which temporarily modulates the signal obtained on the photodetector. Only the object parts that are in the focus have a good contrast and consequently generate stronger signal on the detector. The optically sectioned images are obtained employing a CMOS image detector, which demodulates the time-dependent component of the image. Two different systems for real-time acquisition of optically sectioned images are proposed. The first one is based on a specially designed smart-pixel-detector-array (SPDA), which allows performing the signal processing by an electronic circuit integrated to each pixel of the sensor. The second system utilizes a commercial CMOS image sensor. Here, the images are treated by a digital signal processor (DSP) integrated into the camera (iMVS-155). Both approaches provide real-time acquisition of optically sectioned images. The proof of principle for the new method is demonstrated by imaging artificial three-dimensional reflective samples. The optical sectioning imaging of biological samples in bright-field mode and in fluorescence mode is also demonstrated. The optical sectioning performances of the studied systems are similar to that of the confocal microscope. However the new method has some advantages over the classical confocal system: e.g. the difficulties with the alignment and the post-processing inherent to the confocal system are avoided. The theoretical framework presented in the thesis describes the image formation in structured illumination for both reflective and fluorescent samples. The influence of longitudinal chromatic aberration and spherical aberration caused by specimen induced index mismatch on depth discrimination property are studied. In extension to the work related to optically sectioned microscopy, the quantitative polarization microscopy imaging method is proposed as a new application for CMOS detectors. The polarization state of the illuminating light is dynamically changed and the demodulation detection approach is employed to extract the retardance and azimuth of the birefringent sample in real-time; for a whole field of view. Examples of polarization sensitive measurements for different samples are presented. The theoretical analysis is performed using the Jones formalism. Finally, the potentials and limitations of the CMOS detectors for applications in optical microscopy are discussed. Thanks to constant advancements in CMOS technology, the acquisition speed, resolution and sensitivity of new CMOS detectors generations have been improved. This will allow adapting the methods proposed in this thesis for investigations of fast dynamic process where high temporal and spatial resolution is required.

EPFL2004

DNA biosensor using fluorescence microscopy and impedance spectroscopy

Daniel Berdat, Martinus Gijs

Two types of DNA biosensors are presented. Both sensing principles are demonstrated using synthetic oligomer single-stranded DNA (ssDNA) with concentrations in the micromolar range. A first sensor type is based on the detection of fluorescently labeled ssDNA to a complementary probe that is bound to a silicon substrate by a disuccinimidyl terephtalate and aminosilane immobilization procedure. An enhanced fluorescent response is obtained using constructive interference effects in a fused silica layer deposited before immobilization onto the silicon substrate. The selectivity of different DNA probes towards complementary and non-complementary DNA targets is tested. A second type of DNA sensor is based on the impedimetric response of a solution of unlabeled 20-mer ssDNA in de-ionized water. Interdigitated microelectrodes that are 5 μm wide and separated by 5 μm gaps are microfabricated on glass substrates and the complex impedance of the system in the 100 Hz–100 MHz frequency range is investigated. The proportionality between the measured solution resistance and ssDNA concentration is demonstrated.

2006

Label-free detection of DNA with interdigitated micro-electrodes in a fluidic cell

Daniel Berdat, Martinus Gijs

We investigate the analytical performance of an interdigitated electrode sensor for the label-free detection of DNA, by monitoring the complex impedance of 5 µm wide interdigitated Pt microelectrodes on a glass substrate. We detect the hybridization of unlabeled 38-mer target ssDNA with a complementary probe that is bound on the glass in between the electrodes by a disuccinimidyl terephtalate and aminosilane immobilization procedure. The sensor is mounted in a microfluidic flow cell, in which hybridization is monitored and in situ compared with a reference. After hybridization, the cell is perfused with deionised water and the dependence of the measured conductance due to the immobilized target DNA layer, to target DNA concentrations down to 1 nM is demonstrated. Subsequently, we apply our sensor to the detection of pathogen DNA from Salmonella choleraesuis in dairy food.

2008

Application of CMOS image sensors in optical sectioning and polarization microscopy

Jelena Mitic

EPFL2004