Biosensors based on vertically interrogated optofluidic sensing cells

El objetivo de la presente tesis doctoral es el desarrollo de un nuevo concepto de biosensor óptico sin marcado, basado en una combinación de técnicas de caracterización óptica de interrogación vertical y estructuras sub-micrométricas fabricadas sobre chips de silicio. Las características más importantes de dicho dispositivo son su simplicidad, tanto desde el punto de vista de medida óptica como de introducción de las muestras a medir en el área sensible, aspectos que suelen ser críticos en la mayoría de sensores encontrados en la literatura. Cada uno de los aspectos relacionados con el diseño de un biosensor, que son fundamentalmente cuatro (diseño fotónico, caracterización óptica, fabricación y fluídica/inmovilización química) son desarrollados en detalle en los capítulos correspondientes. En la primera parte de la tesis se hace una introducción al concepto de biosensor, en qué consiste, qué tipos hay y cuáles son los parámetros más comunes usados para cuantificar su comportamiento. Posteriormente se realiza un análisis del estado del arte en la materia, enfocado en particular en el área de biosensores ópticos sin marcado. Se introducen también cuáles son las reacciones bioquímicas a estudiar (inmunoensayos). En la segunda parte se describe en primer lugar cuáles son las técnicas ópticas empleadas en la caracterización: Reflectometría, Elipsometría y Espectrometría; además de los motivos que han llevado a su empleo. Posteriormente se introducen diversos diseños de las denominadas "celdas optofluídicas", que son los dispositivos en los que se va a producir la interacción bioquímica. Se presentan cuatro dispositivos diferentes, y junto con ellos, se proponen diversos métodos de cálculo teórico de la respuesta óptica esperada. Posteriormente se procede al cálculo de la sensibilidad esperada para cada una de las celdas, así como al análisis de los procesos de fabricación de cada una de ellas y su comportamiento fluídico. Una vez analizados todos los aspectos críticos del comportamiento del biosensor, se puede realizar un proceso de optimización de su diseño. Esto se realiza usando un modelo de cálculo simplificado (modelo 1.5-D) que permite la obtención de parámetros como la sensibilidad y el límite de detección de un gran número de dispositivos en un tiempo relativamente reducido. Para este proceso se escogen dos de las celdas optofluídicas propuestas. En la parte final de la tesis se muestran los resultados experimentales obtenidos. En primer lugar, se caracteriza una celda basada en agujeros sub-micrométricos como sensor de índice de refracción, usando para ello diferentes líquidos orgánicos; dichos resultados experimentales presentan una buena correlación con los cálculos teóricos previos, lo que permite validar el modelo conceptual presentado. Finalmente, se realiza un inmunoensayo químico sobre otra de las celdas propuestas (pilares nanométricos de polímero SU-8). Para ello se utiliza el inmunoensayo de albumina de suero bovino (BSA) y su anticuerpo (antiBSA). Se detalla el proceso de obtención de la celda, la funcionalización de la superficie con los bioreceptores (en este caso, BSA) y el proceso de biorreconocimiento. Este proceso permite dar una primera estimación de cuál es el límite de detección esperable para este tipo de sensores en un inmunoensayo estándar. En este caso, se alcanza un valor de 2.3 ng/mL, que es competitivo comparado con otros ensayos similares encontrados en la literatura. La principal conclusión de la tesis es que esta tipología de dispositivos puede ser usada como inmunosensor, y presenta ciertas ventajas respecto a los actualmente existentes. Estas ventajas vienen asociadas, de nuevo, a su simplicidad, tanto a la hora de medir ópticamente, como dentro del proceso de introducción de los bioanalitos en el área sensora (depositando simplemente una gota sobre la micro-nano-estructura). Los cálculos teorícos realizados en los procesos de optimización sugieren a su vez que el comportamiento del sensor, medido en magnitudes como límite de detección biológico puede ser ampliamente mejorado con una mayor compactación de pilares, alcanzandose un valor mínimo de 0.59 ng/mL). The objective of this thesis is to develop a new concept of optical label-free biosensor, based on a combination of vertical interrogation optical techniques and submicron structures fabricated over silicon chips. The most important features of this device are its simplicity, both from the point of view of optical measurement and regarding to the introduction of samples to be measured in the sensing area, which are often critical aspects in the majority of sensors found in the literature. Each of the aspects related to the design of biosensors, which are basically four (photonic design, optical characterization, fabrication and fluid / chemical immobilization) are developed in detail in the relevant chapters. The first part of the thesis consists of an introduction to the concept of biosensor: which elements consists of, existing types and the most common parameters used to quantify its behavior. Subsequently, an analysis of the state of the art in this area is presented, focusing in particular in the area of label free optical biosensors. What are also introduced to study biochemical reactions (immunoassays). The second part describes firstly the optical techniques used in the characterization: reflectometry, ellipsometry and spectrometry; in addition to the reasons that have led to their use. Subsequently several examples of the so-called "optofluidic cells" are introduced, which are the devices where the biochemical interactions take place. Four different devices are presented, and their optical response is calculated by using various methods. Then is exposed the calculation of the expected sensitivity for each of the cells, and the analysis of their fabrication processes and fluidic behavior at the sub-micrometric range. After analyzing all the critical aspects of the biosensor, it can be performed a process of optimization of a particular design. This is done using a simplified calculation model (1.5-D model calculation) that allows obtaining parameters such as sensitivity and the detection limit of a large number of devices in a relatively reduced time. For this process are chosen two different optofluidic cells, from the four previously proposed. The final part of the thesis is the exposition of the obtained experimental results. Firstly, a cell based sub-micrometric holes is characterized as refractive index sensor using different organic fluids, and such experimental results show a good correlation with previous theoretical calculations, allowing to validate the conceptual model presented. Finally, an immunoassay is performed on another typology of cell (SU-8 polymer pillars). This immunoassay uses bovine serum albumin (BSA) and its antibody (antiBSA). The processes for obtaining the cell surface functionalization with the bioreceptors (in this case, BSA) and the biorecognition (antiBSA) are detailed. This immunoassay can give a first estimation of which are the expected limit of detection values for this typology of sensors in a standard immunoassay. In this case, it reaches a value of 2.3 ng/mL, which is competitive with other similar assays found in the literature. The main conclusion of the thesis is that this type of device can be used as immunosensor, and has certain advantages over the existing ones. These advantages are associated again with its simplicity, by the simpler coupling of light and in the process of introduction of bioanalytes into the sensing areas (by depositing a droplet over the micro-nano-structure). Theoretical calculations made in optimizing processes suggest that the sensor Limit of detection can be greatly improved with higher compacting of the lattice of pillars, reaching a minimum value of 0.59 ng/mL)

Label-free Optical Biosensing on a single chip.

Combining semiconductor technology with photonics, optics, and biochemistry leads to sensors with improved biomedical diagnostic capability

Optimization of interferometric photonic cells for biochemical sensing based on advanced high sensitivity optical techniques

The integration between micro-nano fluidics and optics is a new emerging research field, with promising high impact applications in the area of optical biosensing devices. This is the case of tunable Mach-Zehnder interferometers, photonic crystals and ring resonators, which have been demonstrated recently. Recent investigations1 have demonstrated that by combination of the simultaneous used of Ellipsometry, Reflectometry and Spectrometry based technologies broadly used in semiconductor industry at sub-micron spot-size level and advanced photonic structures, a relevant improvement can be achieved at the level of performance of the current state of the art for label-free biosensing and nano-fluidics metrology. The variation in the effective index of refraction can be easily detected in micron/sub-micron domains due to the fact of using several reflectivity profiles and optical responses simultaneously, making possible to remove ambiguities in the sensing interrogation process. To achieve this novel bio-chemical sensing system, it is proposed to combine the miniaturization of sub-micro-holes based Interferometric photonic structures in combination with sub-micron spot size advance optical techniques holistically. Thus, optical sensing system is based on the observation of external reflectivity profile of the high sensitive photonic structures. The reflectivity profile provides four magnitudes which can be used to assess the photonic structures response. These are the reflected amplitude and phase of the electric field components polarized parallel (p) and perpendicular (s) to the plane incidence. The reflected light of the photonic structures produce spectra interference patterns as a function of the angle of incidence for p and s polarization directions and as a function of the spectral range. These patterns are the fundamental source of information to detect the biomolecules binding in the sensing surfaces, ultra small fraction of volumes and flow control in sub-micron domains. Theoretical calculations at CLUPM demonstrate that a detection limit of 10-7 R.I.U. and surface concentration detection limit of 0.1 pg/mm2 are feasible

Integrated slot-waveguide microresonator for biochemical sensing

A novel integrated biochemical sensor based on a slot-waveguide [1] microring resonator is demonstrated. The microresonator is fabricated on a Si3N4/SiO2 material platform [2] by using conventional microfabrication techniques, such as Si thermal oxidation, chemical vapour deposition, electron-beam lithography and reactive ion etching. The sensor consists of a 70-μm-radius ring resonator formed by a slot-waveguide [1] having a slot-width of 200 nm. The operation wavelength is 1.3 μm. The device is exposed to different water-ethanol solutions and its transmission spectrum is measured. A linear shift of the resonant wavelength with increasing ambient refractive index of 212 nm/refractive index units (RIU) is observed. This value is more than twice larger than those of strip-waveguide ring resonator biochemical sensors, indicating that higher analyte-probe light interaction occurs in our slot-waveguide sensor as compared to those based on conventional strip waveguides. The sensor detects a minimal refractive index variation of 2x10-4 RIU, limited by the wavelength resolution of the light source (50 pm). Simulations indicate that the slot region is partially filled when the sensor is exposed to an aqueous solution. We also demonstrate the capability of our sensor to measure higher index fluids such as isopropanol (n=1.37) and cyclohexane (n=1.42)

Characterization of the Performance of Optical Label-Free Biosensors

The field of optical label free biosensors has become a topic of interest during past years, with devices based on the detection of angular or wavelength shift of optical modes [1]. Common parameters to characterize their performance are the Limit of Detection (LOD, defined as the minimum change of refractive index upon the sensing surface that the device is able to detect, and also BioLOD, which represents the minimum amount of target analyte accurately resolved by the system; with units of concentration (common un its are p pm, ng/ml, or nM). LOD gives a first value to compare different biosensors, and is obtained both theoretically (using photonic calculation tools), and experimentally,covering the sensing area with fluids of different refractive indexes

Biosensing comparison between different geometries based on vertical submicron-structrures made of SU-8 resist

Previous work of the research group [1-4] demonstrated the viability of using periodic lattices of micro and nanopillars, called Bio-photonic sensing Cells (BICELLs), as an optical biosensor vertically characterized by visible spectrometry. Also we have studied theoretically [5] the performance of the BICELLs by 2D and 3D simulation in orde r to optimize the biosensing response. In this work we present the fabrication and biosensing comparison of different geometrical parameters on periodic lattices of pillars in order to discuss theoretical conclusions with these results. In this way, we have explored the biosensing response of other patter ns such as crosses, stars, cylinders, concentrical cylinders (Figure 1). Also we introduced a novel method to test the BICELLs in a cost-effective way by using an ultra-thin film of SU-8 spin-coated onto the patterns to reproduce the effect of a biofilm attached to the biosensor surface. Finally we have tested the biosensing response of the different geometries by the well-known Bovine Serum Albumin (BSA) immunoassay and compared with the theoretical simulation

Optimization of Dengue immunoassay by label-free interferometric optical detection method

In this communication we report a direct immunoassay for detecting dengue virus by means of a label-free interferometric optical detection method. We also demonstrate how we can optimize this sensing response by adding a blocking step able to significantly enhance the optical sensing response. The blocking reagent used for this optimization is a dry milk diluted in phosphate buffered saline. The recognition curve of dengue virus over the proposed surface sensor demonstrates the capacity of this method to be applied in Point of Care technology

Metrología óptica dimensional submicrométrica para determinación de espesores en sub-micro estructuras

En este trabajo se presenta el análisis de una técnica de metrología óptica utilizada para el control de procesos en línea en la fabricación de microchips. Se obtienen los perfiles de reflectividad en función del ángulo de incidencia para una longitud de onda de 675 nm, para los estados de polarización s y p. Se obtiene un modelo teórico para una estructura multicapa, con la que se pueden calcular de forma sencilla las propiedades ópticas y dimensiones de las capas. Se obtiene la incertidumbre de la técnica de medida.-In this work is presented the analysis of a technique of optical metrology widely used in controlling on- line process in fabrication of microchips. Reflectivity profiles as a function of angle of incidence are obtained for a wavelength of 675 nm. A theoretical model for a Si/ SiO2 multilayer stack is also obtained, which can be used to calculate both the thickness and the optical properties of the layers. A calculation of the uncertainty for the measurement technique is also performed

Microfabrication processes for microfluidic devices on a single laser Workstation: direct writing lithography on SU-8, laser ablation on polymers and mask manufacturing

We demonstrate the capability of a laser micromachining workstation for cost-effective manufacturing of a variety of microfluidic devices, including SU-8 microchannels on silicon wafers and 3D complex structures made on polyimide Kapton® or poly carbonate (PC). The workstation combines a KrF excimer laser at 248 nm and a Nd3+:YVO4 DPSS with a frequency tripled at 355 nm with a lens magnification 10X, both lasers working at a pulsed regime with nanoseconds (ns) pulse duration. Workstation also includes a high-resolution motorized XYZ-tilt axis (~ 1 um / axis) and a Through The Lens (TTL) imaging system for a high accurate positioning over a 120 x 120 mm working area. We have surveyed different fabrication techniques: direct writing lithography,mask manufacturing for contact lithography and polymer laser ablation for complex 3D devices, achieving width channels down to 13μ m on 50μ m SU-8 thickness using direct writing lithography, and width channels of 40 μm for polyimide on SiO2 plate. Finally, we have tested the use of some devices for capillary chips measuring the flow speed for liquids with different viscosities. As a result, we have characterized the presence of liquid in the channel by interferometric microscopy

Towards In-Vitro Point of Care devices for in-situ diagnosis

Electronic and optoelectronic systems and subsystems play an important role to develop In-Vitro Diagnostics (IVDs) systems for healthcare, clinical, agro-food, environmental, pharmaceutical research or drug control, among many other applications. Although significant advantages have been described for label-free biosensing technology, still a limitednumber of compact devices for monitoring IVD in-situ have been already developed. In this paper a discussion about the current trends for developing Point-of-Care devices will be analyzed, as well as the future challenges for in-situ In-vitro diagnostic systems