La llum a la nanoescala i la seva aplicació a la biomedicina

Conferència realitzada el Dissabte, 13 de Febrer de 2016La llum té una velocitat finita i constant al buit (3×108 m/s), encara que la seva velocitat es redueix en viatjar per un medi ja que interacciona amb la matèria. La ciència que estudia aquesta interacció de la llum amb la matèria és l'òptica. Amb el recent sorgiment de la nanotecnologia, ha estat possible fabricar estructures de mides nanomètriques on la llum, en ser confinada exhibeix propietats extraordinàries. Els fenòmens relacionats amb el confinament de la llum en materials nanoestructurats, han trobat una gran aplicació a l'àrea de la biomedicina i en particular al món del diagnòstic. Mitjançant esquemes altament multidisciplinaris on s'uneixen coneixements relacionats amb la física, la química i la biologia ha estat possible desenvolupar nous dispositius òptics de biosensat. Aquests dispositius ofereixen un anàlisis més ràpid, fiable i simple que les tècniques actuals fent possible la implementació de noves proves diagnòstiques que podrien millorar substancialment l'atenció al pacient (per exemple la detecció precoç i les teràpies personalitzades pel tractament del càncer) o la monitorització mediambiental (detecció "on-line" de contaminants al mar)

Bimodal waveguide interferometer device based on silicon photonics technology for label-free and high sensitive biosensing

Els dispositius òptics biosensors basats en la detecció d'ona evanescent podrien superar les limitacions dels tests de diagnòstic actuals (que són lents i cars) degut a la possibilitat de realitzar les deteccions a temps reals i fent servir un esquema sense la necessitat de marcatges. Entre els diferents transductors òptics, els interferomètrics són els que posseeixen els millors límits de detecció (LOD) deguts a canvis en el índex de refracció de dissolucions (10-7-10-8 Unitats d'Índex de Refracció, RIU) així com per sensibilitat superficial (en el rang dels pg/ml) i un rang lineal més gran. No obstant, les configuracions interferomètriques (l'interferòmetre Mach-Zehnder o el Young) més usuals fan servir un divisor amb forma de Y, que és essencial per dividir o recombinar la llum, lo qual, degut a les toleràncies de les actuals tècniques de fabricació es una gran desavantatge per la reproduibilitat d'aquests dispositius. Per evitar aquests problemes, hem desenvolupat una configuració interferomètrica més simple basada en un guia de ones recte on dos modes de llum de la mateixa polarització interfereixen entre si. Aquesta configuració elimina la complexitat dels interferòmetres més utilitzats i conseqüentment, el biosensors que s'obtenen són més fiables i reproduïbles. Aquesta tesis esta dirigida al desenvolupament i la caracterització d'un nou transductor fotònic per biosensat d'alta sensibilitat i sense marcatges, el dispositiu de guia d'ona bimodal (BiMW). Amb aquest propòsit, els següents punts han estat plantejats: 1.Disseny, fabricació i caracterització òptica del transductor que opera segons el principi de la interferència de dos modes de llum. 2.Desenvolupament i optimització de les estratègies de funcionalització de la superfície transductora fent servir processos de silanització. 3.Estudi de l'aplicabilitat del biosensor amb la demostració del diagnosis analític de problemes clínics rellevants. El transductor es fabrica a nivell d'oblea a la Sala Blanca, lo qual garanteix la producció en massa del dispositiu així com un preu baix del mateix. El dispositiu és molt sensible a variacions en l'índex de refracció de dissolucions, obtenint un límit de detecció de 2×10-7 RIU. La biofuncionalització de l'àrea sensora es un dels aspectes més importants d'aquest treball. Diferents protocols per immobilitzar els diferents bioreceptor en la superfície del dispositiu (cadenes d'ADN, proteïnes i anticossos) han estat desenvolupats. Aquests protocols s'han fet servir per la demostració de diferents bioaplicacions; la detecció d'hormones, bactèries o seqüències d'ADN complementàries. Els resultats presentats en aquesta tesis han destacat pel superior funcionament d'aquest dispositiu en comparació amb els tests de diagnosis convencionals degut a: i) la possibilitat de monitoritzar les interaccions biomoleculars en temps real i fent servir un esquema sense marcadors reduint el temps i el cost de l'assaig, ii) la fabricació del dispositiu fent servir microtecnologia de silici, possibilitant la producció en massa, iii) l'alta sensibilitat (pg/ml, femtomolar) demostrada per les diferents bioaplicacions avaluades i iv) el dispositiu reuneix els requeriments específics per ser miniaturitzat e integrat en una plataforma de sensat multiplexada. Aquest treball obre la porta a la integració d'aquest transductor en un dispositiu lab-on-a -chip, una feina que inclou l'acoblament/detecció de la llum, un sistema capaç de modular la senyal interferomètrica i la incorporació de canals microfluídics per anàlisis multiplexats. Cadascun d'aquests temes afegeix molta complexitat al dispositiu final, han de ser individualment desenvolupades i optimitzades per ser integrades en un biosensor lab-on-a-chip. Finalment, la possibilitat de detectar simultàniament múltiples analits involucra el desenvolupament de noves tècniques per recollir les múltiples senyals així com desenvolupar noves estratègies de biofuncionalització.Optical biosensor devices based on evanescent wave detection could overcome the limitations of conventional diagnostic tests (expensive and time-consuming) due to the possibility of carrying out the detection in real-time and using a label-free scheme. Among the different optical transducers, interferometric devices have evidenced the best limit of detection (LOD) for refractive index changes of bulk solutions (10-7-10-8 Refractive Index Units, RIU) and for surface sensing (in the pg/ml range) and a wider linear range. However, usual interferometric transducers (Mach-Zehnder or Young interferometers) employ the Y-junction to split or recombine light, a drawback for the coherence and performance of the device due to standard tolerances of microfabrication techniques. To overcome these problems, we have developed a simple configuration based on a single straight waveguide where two modes of the light of the same polarization are interfering between them. This simple approach avoids the complexity of the usual interferometric transducers and as a consequence, more reliable and reproducible biosensors can be obtained. This thesis is focused on the development and characterization of a new photonic transducer, the Bimodal Waveguide device (BiMW), for label- free and high sensitive biosensing. To achieve this, the following steps have been pursued: 1. Design, fabrication, and optical characterization of an optical transducer operating by two-mode interference principle. 2. Development and optimization of biofunctionalization strategies on the transducer surface using silanization techniques. 3. Study of the applicability of the biosensor with the demonstration of bioanalytical diagnosis of relevant problems. The transducers are fabricated at wafer level in Clean Room facilities, which warrants a cost-effective and mass-production of the sensor chips. The device is highly sensitive to small changes in the refractive index occurring on the sensor area, leading to a detection limit of 2.5×10-7 RIU for bulk changes in refractive index solutions. The biofunctionalization of the sensor area is one of the most crucial aspects of this work. Optimized functionalization procedures have been achieved, which has been employed to immobilize different types of bioreceptors (DNA strands, proteins, and antibodies) on the surface. The optimized protocols have been used for the demonstration of different bioapplications such as the detection of hormones, bacteria, or complementary DNA sequences. The results presented in this work have highlighted the superior performance of this device in comparison with conventional diagnostics tests due to: i) the possibility of monitoring biomolecular interactions in real-time and by using a label-free scheme which reduce the time and cost of the assay , ii) the fabrication of the device using standard silicon microelectronics technology opening the possibility for mass-production, iii) the high sensitivity demonstrated for the different bioapplications assessed achieving detection limits in the pg/ml range (femtomolar), and iv) the device meets the specific requirements to be miniaturized and integrated in a multiplexed platform. This work opens the door for the integration of this transducer in a lab-on-a-chip device, including the in-coupling/out-coupling of light, a system able to modulate the interferometric signal, and the incorporation of microfluidics channels for multiplexing. Each of these subjects adds a great complexity to the final device, and must be independently developed and optimized in order to be successfully integrated at the final lab-on-a-chip biosensor. Finally, the possibility to detect simultaneously multiple analytes will involve further efforts in developing new optical in and outcoupling as well as new biofunctionalization strategies

Label-free detection of nosocomial bacteria using a nanophotonic interferometric biosensor

Nosocomial infections are a major concern at the worldwide level. Early and accurate identification of nosocomial pathogens is crucial to provide timely and adequate treatment. A prompt response also prevents the progression of the infection to life-threatening conditions, such as septicemia or generalized bloodstream infection. We have implemented two highly sensitive methodologies using an ultrasensitive photonic biosensor based on a bimodal waveguide interferometer (BiMW) for the fast detection of Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus (MRSA), two of the most prevalent bacteria associated with nosocomial infections. For that, we have developed a biofunctionalization strategy based on the use of a PEGylated silane (silane-PEG-COOH) which provides a highly resistant and bacteria-repelling surface, which is crucial to specifically detect each bacterium. Two different biosensor assays have been set under standard buffer conditions: One based on a specific direct immunoassay employing polyclonal antibodies for the detection of P. aeruginosa and another one employing aptamers for the direct detection of MRSA. The biosensor immunoassay for P. aeruginosa is fast (it only takes 12 min) and specific and has experimentally detected concentrations down to 800 cfu mL (cfu: Colony forming unit). The second one relies on the use of an aptamer that specifically detects penicillin-binding protein 2a (PBP2a), a protein only expressed in the MRSA mutant, providing a photonic biosensor with the ability to identify the resistant pathogen MRSA and differentiate it from methicillin-susceptible S. aureus (MSSA). Direct, label-free, and selective detection of whole MRSA bacteria has been achieved, making possible the direct detection of also 800 cfu mL. According to the signal-to-noise (S/N) ratio of the device, a theoretical limit of detection (LOD) of around 49 and 29 cfu mL was estimated for P. aeruginosa and MRSA, respectively. Both results obtained under standard conditions reveal the great potential this interferometric biosensor device has as a versatile and specific tool for bacterial detection and quantification, providing a rapid method for the identification of nosocomial pathogens within the clinical requirements of sensitivity for the diagnosis of infections

Integrated bimodal waveguide interferometric biosensor for label-free analysis

The performance of an interferometric device based on integrated Bimodal Waveguides (BiMW) for sensing is demonstrated. The sensors are fabricated using standard silicon technology and can achieve a detection limit of 2.5 · 10 RIU for homogeneous sensing, rendering in a very high sensitive device. The applicability of the bimodal waveguide interferometer as label-free biosensor has been demonstrated by the real-time monitoring of the biomolecular interaction of BSA and antiBSA. Due to their simplicity, the interferometric devices could be further integrated in complete lab-on-a-chip platforms for point-of-care diagnostics showing them as a powerful instrument for biochemical analysis

Label-free bimodal waveguide immunosensor for rapid diagnosis of bacterial infections in cirrhotic patients

Spontaneous bacterial peritonitis is an acute bacterial infection of ascitic fluid; it has a high incidence in cirrhotic patients and it is associated with high mortality. In such a situation, early diagnosis and treatment is crucial for the survival of the patient. However, bacterial analysis in ascitic fluid is currently based on culture methods, which are time-consuming and laborious. We report here the application of a photonic interferometer biosensor based on a bimodal waveguide (BiMW) for the rapid and label-free detection of bacteria directly in ascitic fluid. The device consists of a straight waveguide in which two modes of the same polarization interfere while interacting with the external medium through their evanescent fields. A bimolecular event occurring on the sensor area of the device (e.g. capturing bacteria) will differently affect each light mode, inducing a variation in the phase of the light exiting at the output of the waveguide. In this work, we demonstrate the quantitative detection of Bacillus cereus in buffer medium and Escherichia coli in undiluted ascitic fluid from cirrhotic patients. In the case of Bacillus cereus detection, the device was able to specifically detect bacteria at relevant concentrations in 12.5 min and in the case of Escherichia coli detection, the analysis time was 25 min. Extrapolation of the data demonstrated that the detection limits of the biosensor could reach few bacteria per milliliter. Based on the results obtained, we consider that the BiMW biosensor is positioned as a promising new clinical tool for user-friendly, cost-effective and real-time microbiological analysis

Trends in photonic lab-on-chip interferometric biosensors for point-of-care diagnostics

Portable point-of care (POC) devices for in vitro diagnostics will be a milestone for the achievement of universal healthcare and environmental protection. The main goal is to reach a rapid, user-friendly and highly sensitive portable tool which can provide immediate results in any place at any time while having a competitive cost. Integrated optical (IO) waveguide based-biosensors are the most suitable candidates to achieve this ambitious objective. They are able to operate in real samples (such as blood, urine, wastewater…) affording relevant sensitivities even under a label-free scheme. In addition, arrays of IO sensors for multiplexed analysis can be integrated in lab-on-chip (LOC) platforms, providing a truly cost-effective fabrication and miniaturization. Among the different IO biosensors, interferometric ones have demonstrated the highest sensitivity for label-free detection ever reported. Although the first interferometric biosensors were developed in the early nineties, they focused mainly on preliminary proof-of-concept studies; only recently the resilient potential of interferometric biosensors as highly advanced POC devices has firmly emerged. This review provides an overview of the state-of-the art in photonic interferometric biosensors, their main biofunctionalisation routes and their integration in LOC platforms, while maintaining a special focus on the real analytical applications achieved so far

Impact of the genetic improvement of fermenting yeasts on the organoleptic properties of beer

The brewing industry has experienced a significant boom in recent years through the emergence of, on the one hand, craft breweries that produce beers with unique organoleptic characteristics, and, on the other hand, the brewing of a significant number of beers using hybridized or genetically modified microorganisms with the aim of improving both the brewing processes and the final products. This review covers the influence from yeast strains on the organoleptic properties of the final beers and also the main hybridization and genetic modification methods applied to such yeast strains with the aim of improving the sensory characteristics of the product obtained and/or the brewing process. Different approaches to the phenotypic modification of the yeasts used in beer brewing have arisen in recent years. These are dealt with in this work, with special emphasis on the methodology followed as well as on the effects of the same on the brewing process and/or on the final produc

Linear readout of integrated interferometric biosensors using a periodic wavelength modulation

An all-optical phase modulation method for the linear readout of integrated interferometric biosensors is demonstrated, merging simple intensity detection with the advantages offered by spectral interrogation. The phase modulation is introduced in a simple and cost-effective way by tuning a few nanometers the emission wavelength of commercial laser diodes, taking advantage of their well-known drawback of power-wavelength dependence. The method is applied to the case of a bimodal waveguide (BiMW) interferometric biosensor, fabricated with standard silicon technology and operated at visible wavelengths, rendering a detection limit of 4×10-7 refractive index units for bulk sensing. The biosensing capabilities of the phase-linearized BiMW device are assessed through the quantitative immunoassay of C-reactive protein, a key protein in inflammatory processes. This method can be applied to any modal interferometer. To solve the ambiguities affecting interferometric biosensors, a phase modulation system based on variations of the incident wavelength and Fourier deconvolution is presented. The wavelength variation is introduced taking advantage of the power-wavelength dependence of commercial laser diodes, resulting in a cost-effective method, valid for all modal interferometers. Considering the modulation of a bimodal waveguide interferometric sensor, limits of detection of 4 · 10 for bulk sensing and 7 ng/ml for the detection of C-Reactive protein were demonstrated