Plasmonic-based impedance microspectroscopy of optically heterogeneous samples

A robust impedance microscopy technique is presented. This optical tool enables high resolution imaging of electrical properties with promising biophysical applications. The underlying principle is that surface plasmon resonance (SPR) sensors are able to measure perturbations of surface charge density and therefore can be used to compute the impedance of surface-adhered cells. However, the ability to perform reliable quantitative impedance imaging is affected by the optical heterogeneity of the cell-sensor interface. To address this issue, a novel method for quantitative time-resolved resonance angle tracking is developed and applied to correct for the effect of the optical properties. To demonstrate the capability of this technique, impedance microspectroscopy of bovine serum albumin (BSA) patterns was performed enabling measurements of capacitance with submicroscopic resolution. The work presented offers an impedance microspectroscopy method that will create new avenues in studying the electrical properties of single cells and biomolecules as well as bio-electrical currents

Sensing voltage dynamics with differential intensity surface plasmon resonance system

The voltage sensitivity of surface plasmon resonance is investigated as a potential method for label-free detection of the dynamics of the transmembrane potential of excitable cells. Development of such a method for reliable detection of these signals is one of the modern challenges in biomedical research. Since they are key physiological signals that control a number of vital functions, they report the system's behaviour in health and disease. Labelling methods are currently used to detect these signals, often with subcellular resolution, but fluorescent labels (i) have a short lifetime, which limits the timescale of the experiments and (ii) they can be toxic to cells. Surface plasmon resonance (SPR) is sensitive to perturbation of the potential at the metal-electrolyte interface. This perturbation alters the charge density of the interface, which is characterised by the well-known theory of the double-layer capacitance. Charge density on the electrolyte side is mirrored by an excess or deficiency of electrons at the metal surface. The latter shifts the resonance conditions of surface plasmons. To assess SPR capacity in the detection of the electrical signals from excitable cells, the limit of voltage detection of the SPR has to be well-characterised. First, theoretical approaches were used to estimate the voltage detection limit of surface plasmon resonance. Since different SPR systems are characterised by their refractive index sensitivity, voltage sensitivity was estimated relative to the equivalent refractive index change. This approach enables the generalisation of the outputs of this research regardless of the SPR system. A one-dimensional multilayer model was used combining the electrical properties of the metal-electrolyte interface to the optical properties of the layers. The model was used to calculate the voltage-induced changes to the optical properties of the metal surface. Then, light reflectivity from the model structure was calculated using the transfer matrix of the structure and Fresnel equations to produce SPR curves (reflectance versus angle of incidence) for a series of voltages in the double-layer charging range (_200mV). Second, to test the theoretical estimates of the voltage sensitivity of surface plasmon resonance, a differential-intensity surface plasmon resonance (DI-SPR) system was constructed and combined with an electrochemical system to control the potential at the metal-electrolyte interface. The detection limit of the system is in the range of 3 _ 1

Characterization of candidate genes in unexplained polyposis and colorectal cancer

Approximately 35% of colorectal cancer (CRC) risk is attributed to heritable factors, with 5 to 10% linked to dominant or recessive inherited syndromes. Known high-risk genes like POLE, POLD1, NTHL1 and APC contribute to a portion of this risk. However, the genetic basis for 20%-30% of inherited CRC remains unclear. This thesis explores the roles of POLE, POLD1, APC and NTHL1 in CRC and polyposis. While screening for pathogenic variants in POLE and POLD1, remarkably POLE L424V variants were found to induce Lynch syndrome-like features due to somatic mismatch repair gene mutations. Biallelic NTHL1 variants predisposing to CRC and polyposis were studied in a collaborative effort, describing a broad tumor spectrum and a high risk of extracolonic cancers associated with NTHL1 deficiency. For monoallelic NTHL1 variant carriers, no significant evidence link was found with increased polyposis or CRC risk, as supported by mutational signature analysis on colorectal tumors.LUMC / Geneeskund

Analysis of noise in differential and ratiometric biosensing systems

This paper presents formulations to evaluate noise in differential and ratiometric measurements that are often performed in biosensing. These measurements are performed to improve signal to noise ratio of the sensing systems for sensitive detection of dynamic biological processes. The use of these formulations is discussed in the context of the differential intensity surface plasmon resonance (SPR) system that is widely used to characterise molecular interactions on a confined axial scale. Previous studies provide qualitative descriptions of the noise performance of such systems but lack rigorous characterisation. Here we present analytical expressions for quantitative evaluation of the noise in differential and ratiometric measurements by applying the rules of arithmetic operations on random variables. Such formulations provide the means for evaluating the signal to noise ratio of such systems. We present how correlated noise can be removed by performing differential or ratiometric processing. Applying these formulations, we also show how the sensitivity of the differential intensity SPR system changes during the experiment

Impedimetric Characterization of Bipolar Nanoelectrodes with Cancer Cells

Merging of electronics with biology, defined as bioelectronics, at the nanoscale holds considerable promise for sensing and modulating cellular behavior. Advancing our understanding of nanobioelectronics will facilitate development and enable applications in biosensing, tissue engineering, and bioelectronic medicine. However, studies investigating the electrical effects when merging wireless conductive nanoelectrodes with biology are lacking. Consequently, a tool is required to develop a greater understanding of merging conductive nanoparticles with cells. Herein, this challenge is addressed by developing an impedimetric method to evaluate bipolar electrode (BPE) systems that could report on electrical input. A theoretical framework is provided, using impedance to determine if conductive nanoparticles can be polarized and used to drive current. It is then demonstrated that 125 nm of gold nanoparticle (AuNP) bipolar electrodes (BPEs) could be sensed in the presence of cells when incorporated intracellularly at 500 μg/mL using water and phosphate-buffered saline (PBS) as electrolytes. These results highlight how nanoscale BPEs act within biological systems. This research will impact the rational design of using BPE systems in cells for both sensing and actuating applications

Sensitive detection of voltage transients using differential intensity surface plasmon resonance system

This paper describes theoretical and experimental study of the fundamentals of using surface plasmon resonance (SPR) for label-free detection of voltage. Plasmonic voltage sensing relies on the capacitive properties of metal-electrolyte interface that are governed by electrostatic interactions between charge carriers in both phases. Externally-applied voltage leads to changes in the free electron density in the surface of the metal, shifting the SPR position. The study shows the effects of the applied voltage on the shape of the SPR curve. It also provides a comparison between the theoretical and experimental response to the applied voltage. The response is presented in a universal term that can be used to assess the voltage sensitivity of different SPR instruments. Finally, it demonstrates the capacity of the SPR system in resolving dynamic voltage signals; a detection limit of 10mV with a temporal resolution of 5ms is achievable. These findings pave the way for the use of SPR systems in the detection of electrical activity of biological cells

Surface plasmon resonance imaging of excitable cells

Surface plasmons (SPs) are surface charge density oscillations occuring at a metal/dieletric interface and are highly sensitive to refractive index variations adjacent to the surface. This sensitivity has been exploited successfully for chemical and biological assays. In these systems, a surface plasmon resonance (SPR)-based sensor detects temporal variations in the refractive index at a point. SPR has also been used in imaging systems where the spatial variations of refractive index in the sample provide the contrast mechanism. SPR imaging systems using high numerical aperture (NA) objective lenses have been designed to image adherent live cells with high magnification and near-diffraction limited spatial resolution. Addressing research questions in cell physiology and pharmacology often requires the development of a multimodal microscope where complementary information can be obtained.In this paper, we present the development of a multimodal microscope that combines SPR imaging with a number of additional imaging modalities including bright-field, epifluorescence, total internal reflection microscopy and SPR fluorescence microscopy. We used a high NA objective lens for SPR and TIR microscopy and the platform has been used to image live cell cultures demonstrating both fluorescent and label-free techniques. Both the SPR and TIR imaging systems feature a wide field of view (~300 µm) that allows measurements from multiple cells whilst maintaining a resolution sufficient to image fine cellular processes. The capability of the platform to perform label-free functional imaging of living cells was demonstrated by imaging the spatial variations in contractions from stem cell-derived cardiomyocytes. This technique shows promise for non-invasive imaging of cultured cells over very long periods of time during development

Responsivity of the differential-intensity surface plasmon resonance instrument

Surface plasmon resonance is used for the sensitive measurement of minute concentrations of bio-analytes and probing of electrochemical processes. Typical refractive index sensitivity, for the intensity approach, is around 10−6 refractive index units (RIUs). A better sensitivity has been suggested by developing a differential-intensity detection method. This method relies on the excitation of surface plasmons using a weakly focused beam with the average angle of incidence equal to the resonance angle, while the reflected light is detected using a bi-cell photodiode. The Bi-cell signal is processed by calculating the difference between its two units, normalized to their sum. This ratio estimates the shift in the resonance angle using a model that represents the resonance curve with a quadratic function. However, this model does not explain the effects of parameters such as the angular width of the excitation beam and the specifications of the sensing structure on the system’s response. This paper presents a detailed evaluation of the responsivity using experimental and theoretical approaches, which can predict the effect of the different parameters, paving the way towards the investigation of a better sensitivity and the optimization of the system’s design for different applications

Wireless bioelectronic nanosystems for intracellular communication

In order for the field of bioelectronics to make an impact on healthcare, there is an urgent requirement for the development of “wireless” electronic systems to both sense and actuate cell behaviour. Herein we report the first example of an innovative intracellular wireless electronic communication system. We demonstrate that chemistry can be electrically modulated in a “wireless” manner on the nanoscale at the surface of conductive nanoparticles uptaken by cells at unreported low potentials. The system is made functional by modifying gold nanoparticles incorporating a Zn-porphyrin, which are taken up by cells and are shown to be biocompatible. It is demonstrated the redox state of Zn-porphyrin modified gold nanoparticles is modulated and reported on fluorescently when applying an external electrical potential. This provides an attractive new “wireless” approach to develop novel bioelectronic devices for modulating and sensing cellular behaviour using intracellular monitoring

Label-free Brillouin endo-microscopy for the quantitative 3D imaging of sub-micrometre biology

This report presents an optical fibre-based endo-microscopic imaging tool that simultaneously measures the topographic profile and 3D viscoelastic properties of biological specimens through the phenomenon of time-resolved Brillouin scattering. This uses the intrinsic viscoelasticity of the specimen as a contrast mechanism without fluorescent tags or photoacoustic contrast mechanisms. We demonstrate 2 µm lateral resolution and 320 nm axial resolution for the 3D imaging of biological cells and Caenorhabditis elegans larvae. This has enabled the first ever 3D stiffness imaging and characterisation of the C. elegans larva cuticle in-situ. A label-free, subcellular resolution, and endoscopic compatible technique that reveals structural biologically-relevant material properties of tissue could pave the way toward in-vivo elasticity-based diagnostics down to the single cell level