Numa Couniot

Lytic enzymes as selectivity means for label-free, microfluidic and impedimetric detection of whole-cell bacteria using ALD-Al2O3 passivated microelectrodes

Point-of-care (PoC) diagnostics for bacterial detection offer tremendous prospects for public health care improvement. However, such tools require the complex combination of the following performances: rapidity, selectivity, sensitivity, miniaturization and affordability. To meet these specifications, this paper presents a new selectivity method involving lysostaphin together with a CMOS-compatible impedance sensor for genus-specific bacterial detection. The method enables the sample matrix to be directly flown on the polydopamine-covered sensor surface without any pre-treatment, and considerably reduces the background noise. Experimental proof-of-concept, explored by simulations and confirmed through a setup combining simultaneous optical and electrical real-time monitoring, illustrates the selective and capacitive detection of Staphylococcus epidermidis in synthetic urine also containing Enterococcus faecium. While providing capabilities for miniaturization and system integration thanks to CMOS compatibility, the sensors show a detection limit of ca. 10(8) (CFU/mL).min in a 1.5 μL microfluidic chamber with an additional setup time of 50 min. The potentials, advantages and limitations of the method are also discussed.

A 16

We present a 16 × 16 CMOS biosensor array aiming at impedance detection of whole-cell bacteria. Each 14 μm×16 μm pixel comprises high-sensitive passivated microelectrodes connected to an innovative readout interface based on charge sharing principle for capacitance-to-voltage conversion and subthreshold gain stage to boost the sensitivity. Fabricated in a 0.25 μm CMOS process, the capacitive array was experimentally shown to perform accurate dielectric measurements of the electrolyte up to electrical conductivities of 0.05 S/m, with maximal sensitivity of 55 mV/fF and signal-to-noise ratio (SNR) of 37 dB. As biosensing proof of concept, real-time detection of Staphylococcus epidermidis binding events was experimentally demonstrated and provides detection limit of ca. 7 bacteria per pixel and sensitivity of 2.18 mV per bacterial cell. Models and simulations show good matching with experimental results and provide a comprehensive analysis of the sensor and circuit system. Advantages, challenges and limits of the proposed capacitive biosensor array are finally described with regards to literature. With its small area and low power consumption, the present capacitive array is particularly suitable for portable point-of-care (PoC) diagnosis tools and lab-on-chip (LoC) systems.

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To enable impedimetric sensing of whole-cell bacteria in a saline buffer with passivated microelectrodes, we designed a very high frequency (up to 575 MHz) complementary meta-oxide-semiconductor (CMOS) oscillator connected to on-chip Al/Al2O3 interdigitated microelectrodes. The output frequency was theoretically and experimentally shown to be inversely proportional to the permittivity of the solution with a maximal error of 2%, up to an electrical conductivity of 1 S/m. As a biosensing proof-of-concept, successful experimental detection of whole-cell Staphylococcus epidermidis was performed in physiological buffers, showing a detection limit of 107 CFU/ml after 20 min of bacterial incubation. The 0.25-μm CMOS circuit, combining sensing/readout parts, has a 0.05 mm2 area, consumes 29 mW at 250 kS/s, and can be connected to microcontrollers, making the system suitable for point-of-care diagnostics.

16 CMOS Capacitive Biosensor Array Towards Detection of Single Bacterial Cell

We propose a CMOS image sensor operating at ultra-low voltage (ULV) in a 65-nm low-power (LP) CMOS logic process for ultra-low-power SoC integration. Energy of 17-pJ/frame.pixel and 4×4-μm pixel size with 57-% fill factor are achieved at 0.5 V with digital pixel sensor (DPS) and time-based readout while reaching 40-dB dynamic range (DR) despite high leakage currents and Vt variability, thanks to delta-reset sampling (DRS) as well as gating and adaptive body biasing (ABB) of the 2-transistor (2-T) in-pixel comparator.

A Capacitance-to-Frequency Converter With On-Chip Passivated Microelectrodes for Bacteria Detection in Saline Buffers Up to 575 MHz

Adding vision capabilities to wireless sensors nodes (WSN) for the Internet-of-Things requires imagers working at ultra-low power (ULP) in nanometer CMOS systems-on-chip (SoCs). Such performance can be obtained with time-based digital pixel sensors (DPS) working at ultra-low voltage (ULV), at the expense of lower dynamic range, higher fixed-pattern noise (FPN) and thus poorer image quality. To address this problem, three key techniques were developed in this work for DPS pixels: wide-range adaptive body biasing, low-gating of the 2-transistor in-pixel comparator and digital readout performing delta-reset sampling with low switching activity and robust timing closure. These concepts were demonstrated by designing and fabricating a 128 × 128 CMOS image sensor array in a 65 nm low-power CMOS logic process. Operating at 0.5 V, it features an FPN of 0.66%, a dynamic range of 42 dB and a fill factor of 57% with a 4 μm pixel pitch, while consuming only 17 pJ/(frame.pixel) and 8.8 μW at 32 fps. These performances combined with the small silicon area of 0.69 mm2 makes the imager perfectly suitable for integration in ULP SoCs, targeting WSN applications.

A 65-nm 0.5-V 17-pJ/frame.pixel DPS CMOS image sensor for ultra-low-power SoCs achieving 40-dB dynamic range

The specificity of biosensors is typically obtained by surface biofunctionalization, which enables the selective binding of biomolecules. This critical step is sensitive to the nature of materials and to the overall experimental conditions. Here, we provide a comprehensive study of several biofunctionalization methods, including the layer-by-layer technique and both the gas-phase and liquid-phase silanizations, and we propose a new maleimide-based protocol for grafting a protein to a sensor covered by alumina. This method was then validated by making a respiratory syncitial virus-specific biosensor.

A 65 nm 0.5 V DPS CMOS Image Sensor With 17 pJ/Frame.Pixel and 42 dB Dynamic Range for Ultra-Low-Power SoCs

Cell aggregation plays a key role in biofilm formation and pathogenesis of Staphylococcus species. Although the molecular basis of aggregation in Staphylococci has already been extensively investigated, the influence of environmental factors, such as ionic strength, remains poorly understood. In this paper, we report a new type of cellular aggregation of Staphylococci that depends solely on ionic strength. Seven strains out of 14, all belonging to staphylococcal species, formed large cell clusters within minutes in buffers of ionic strength ranging from 1.5 to 50 mM, whereas isolates belonging to other Gram-positive species did not display this phenotype. Atomic force microscopy (AFM) with chemically functionalized tips provided direct evidence that ionic strength modulates cell surface adhesive properties through changes in cell surface charge. The optimal ionic strength for aggregation was found to be strain dependent, but in all cases, bacterial aggregates formed at an ionic strength of 1.5-50 mM were rapidly dispersed in a solution of higher ionic strength, indicating a reversibility of the cell aggregation process. These findings suggest that some staphylococcal isolates can respond to ionic strength as an external stimulus to trigger rapid cell aggregation in a way that has not yet been reported.

Assessment of different functionalization methods for grafting a protein to an alumina-covered biosensor

Capacitive biosensors are promising tools toward detection of bacterial cells. However, their sensitivity remains insufficient compared with more conventional techniques. To understand how to optimize it, we propose a complete and quantitative analysis of the sensitivity of capacitive biosensors using a previously developed 2-D numerical simulator based on Poisson-Nernst-Planck equations. All key parameters of the electrolyte, bacterial cell, and electrode design are investigated, studied, and optimized. For each, an analytical expression relating the sensitivity to the parameter of interest is given. The guidelines and design tools, provided throughout this paper for the designer of capacitive biosensors, are eventually demonstrated by improving the sensitivity of our device under test by a factor of 4. Discussions based on experimental values and analytical models are provided throughout this paper.