Cellular sensing platform with enhanced sensitivity based on optogenetic modulation of cell homeostasis.

We demonstrate a new biosensing concept with impact on the development of rapid, point of need cell based sensing with boosted sensitivity and wide relevance for bioanalysis. It involves optogenetic stimulation of cells stably transfected to express light sensitive protein channels for optical control of membrane potential and of ion homeostasis. Time-lapse impedance measurements are used to reveal cell dynamics changes encompassing cellular responses to bioactive stimuli and optically induced homeostasis disturbances. We prove that light driven perturbations of cell membrane potential induce homeostatic reactions and modulate transduction mechanisms that amplify cellular response to bioactive compounds. This allows cell based biosensors to respond more rapidly and sensitively to low concentrations of bioactive/toxic analytes: statistically relevant impedance changes are recorded in less than 30 min, in comparison with >8 h in the best alternative reported tests for the same low concentration (e.g. a concentration of 25 µM CdCl2, lower than the threshold concentration in classical cellular sensors). Comparative analysis of model bioactive/toxic compounds (ouabain and CdCl2) demonstrates that cellular reactivity can be boosted by light driven perturbations of cellular homeostasis and that this biosensing concept is able to discriminate analytes with different modes of action (i.e. CdCl2 toxicity versus ion pump inhibition by ouabain), a significant advance against state of the art cell based sensors.

High speed CMOS acquisition system based on FPGA embedded image processing for electro-optical measurements.

Electro-optical measurements, i.e., optical waveguides and plasmonic based electrochemical impedance spectroscopy (P-EIS), are based on the sensitive dependence of refractive index of electro-optical sensors on surface charge density, modulated by an AC electrical field applied to the sensor surface. Recently, P-EIS has emerged as a new analytical tool that can resolve local impedance with high, optical spatial resolution, without using microelectrodes. This study describes a high speed image acquisition and processing system for electro-optical measurements, based on a high speed complementary metal-oxide semiconductor (CMOS) sensor and a field-programmable gate array (FPGA) board. The FPGA is used to configure CMOS parameters, as well as to receive and locally process the acquired images by performing Fourier analysis for each pixel, deriving the real and imaginary parts of the Fourier coefficients for the AC field frequencies. An AC field generator, for single or multi-sine signals, is synchronized with the high speed acquisition system for phase measurements. The system was successfully used for real-time angle-resolved electro-plasmonic measurements from 30 Hz up to 10 kHz, providing results consistent to ones obtained by a conventional electrical impedance approach. The system was able to detect amplitude variations with a relative variation of ±1%, even for rather low sampling rates per period (i.e., 8 samples per period). The PC (personal computer) acquisition and control software allows synchronized acquisition for multiple FPGA boards, making it also suitable for simultaneous angle-resolved P-EIS imaging.

Quantitative assessment of specific carbonic anhydrase inhibitors effect on hypoxic cells using electrical impedance assays.

Carbonic anhydrase IX (CA IX) is an important orchestrator of hypoxic tumour environment, associated with tumour progression, high incidence of metastasis and poor response to therapy. Due to its tumour specificity and involvement in associated pathological processes: tumourigenesis, angiogenesis, inhibiting CA IX enzymatic activity has become a valid therapeutic option. Dynamic cell-based biosensing platforms can complement cell-free and end-point analyses and supports the process of design and selection of potent and selective inhibitors. In this context, we assess the effectiveness of recently emerged CA IX inhibitors (sulphonamides and sulphocoumarins) and their antitumour potential using an electrical impedance spectroscopy biosensing platform. The analysis allows discriminating between the inhibitory capacities of the compounds and their inhibition mechanisms. Microscopy and biochemical assays complemented the analysis and validated impedance findings establishing a powerful biosensing tool for the evaluation of carbonic anhydrase inhibitors potency, effective for the screening and design of anticancer pharmacological agents.

Biosensing Based on Magneto-Optical Surface Plasmon Resonance.

In spite of the high analytic potential of Magneto Optical Surface Plasmon Resonance (MOSPR) assays, their applicability to biosensing has been limited due to significant chip stability issues. We present novel solutions to surpass current limitations of MOSPR sensing assays, based on innovative chip structure, tailored measurements and improved data analysis methods. The structure of the chip is modified to contain a thin layer of Co-Au alloy instead of successive layers of homogenous metals with magnetic and plasmonic properties, as currently used. This new approach presents improved plasmonic and magnetic properties, yet a structural stability similar to standard Au-SPR chips, allowing for bioaffinity assays in saline solutions. Moreover, using a custom-designed measurement configuration that allows the acquisition of the SPR curve, i.e., the reflectivity measured at multiple angles of incidence, instead of the reflectivity value at a single-incidence angle, a high signal-to-noise ratio is achieved, suitable for detection of minute analyte concentrations. The proposed structure of the MOSPR sensing chip and the procedure of data analysis allow for long time assessment in liquid media, a significant advancement over existing MOSPR chips, and confirm the MOSPR increased sensitivity over standard SPR analyses.

Assessment of pathogenic bacteria using periodic actuation.

A new analytical platform for the assessment of pathogenic bacteria is presented. It is based on a robust technology which is able to amplify the signal to noise ratio providing fast and sensitive detection of target pathogenic bacteria. The system uses a custom made AC electrical impedance analyser to measure, using a lab on a chip platform, the oscillations of magnetically labelled analytes when applying a periodic magnetic field. The concentration of pathogenic Escherichia coli O157:H7 chosen as bacterial model was determined based on the amplitude of the electrical impedance oscillations at a selected AC frequency. The analytical platform provides a limit of detection of 10(2) cells ml(-1), has a fast analysis time, and is amenable for the detection of other target cells. The system has simple design suitable for portability and automated operation.