Biomolecule recognition using piezoresistive nanomechanical force probes

Highly sensitive sensors are one of the enabling technologies for the biomarker detection in early stage diagnosis of pathologies. We have developed a self-sensing nanomechanical force probe able for detecting the unbinding of single couples of biomolecular partners in nearly physiological conditions. The embedding of a piezoresistive transducer into a nanomechanical cantilever enabled high force measurement capability with sub 10-pN resolution. Here, we present the design, microfabrication, optimization, and complete characterization of the sensor. The exceptional electromechanical performance obtained allowed us to detect biorecognition specific events underlying the biotin-avidin complex formation, by integrating the sensor in a commercial atomic force microscope.This work has been supported by the Spanish Ministry of Science and Innovation through projects NANOSELECT-CSD2007-00041(Consolider-Ingenio 2010) and TEC2011-23600, by the European Union through the COST ACTION TD1002 and partly supported by the PRIN-MIUR Project No. 2009 WPZM4S and by AIRC (Grant IG10412.)Peer reviewe

Fast on-wafer electrical, mechanical, and electromechanical characterization of piezoresistive cantilever force sensors

Validation of a technological process requires an intensive characterization of the performance of the resulting devices, circuits or systems. The technology for the fabrication of Micro and Nanoelectromechanical systems is evolving rapidly, with new kind of device concepts for applications like sensing or harvesting are being proposed and demonstrated. However, the characterization tools and methods for these new devices are still nor fully developed. Here, we present an on-wafer, highly precise and rapid characterization method to measure the mechanical, electrical and electromechanical properties of piezoresistive cantilevers. The set-up is based on a combination of probe-card and atomic force microscopy (AFM) technology, it allows accessing many devices across a wafer and it can be applied to a broad range of MEMS and NEMS. Using this set-up we have characterized the performance of multiple submicron thick piezoresistive cantilever force sensors. For the best design we have obtained a force sensitivity RF=158 uV/nN, a noise of 5.8 uV (1Hz-1kHz) and a minimum detectable force (MDF) of 37 pN with a relative standard deviation of sigma=8%. This small value of sigma, together with a high fabrication yield >95%, validates our fabrication technology. The devices are intended to be used as bio-molecular detectors for the measurement of intermolecular forces between ligand and receptor molecule pairs.This work has been supported by MICINN through projects TEC2011-23600 and NANOSELECT-CSD2007- 00041 (Consolider-Ingenio 2010 Programme).Peer reviewe

Optimization of the force and power consumption of a microfabricated magnetic actuator

The force (F) and the power consumption (P ) of a magnetic actuator are modeled, measured and optimized in the context of developing micro-actuators for large arrays, such as in portable tactile displays for the visually impaired. We present a novel analytical approach complemented with finite element simulation (FEM) and experiment validation, showing that the optimization process can be performed considering a single figure of merit. The magnetic actuator is a disc-shaped permanent magnet displaced by planar microcoil. Numerous design parameters are evaluated, including the width and separation of the coil traces, the trace thickness, number of turns and the maximum and minimum radius of the coil. We obtained experimental values ranging from 2 to 12 mN/ sqrt(W) using up to 2-layer coils of both microfabricated and commercial printed circuit board (PCB) technologies. This performance can be further improved by a factor of two by adopting a 6-layer technology. The method can be applied to a wide range of electromagnetic actuators

Force sensors based on piezoresistive and MOSFET cantilevers for biomolecular sensing

Los procesos de reconocimiento biomolecular entre receptores y ligandos son muy importantes en biología. Estas biomoléculas pueden desarrollar complejos muy específicos y tener una variedad de funciones como replicación y transcripción genómica, actividad enzimática, respuesta inmune, señalamiento celular, etc. La complementariedad inequívoca mostrada por estos componentes biológicos es ampliamente utilizada para desarrollar biosensores. Dependiendo de la naturaleza de las señales que se convierten, los biosensores pueden ser clasificados en ópticos, eléctricos o mecánicos. Entre los sensores mecánicos, los microcantilevers son los más comunes. Han sido utilizados como sensores de estrés superficial o como sensores de masa en detección de biomoléculas, desde hace más de 10 años. El enlace de las moléculas a sus superficies funcionalizadas se puede detectar midiendo la deflexión en modo estático o la variación de la frecuencia de resonancia en modo dinámico. Para lograr la máxima resolución, la deflexión es medida por un láser y un fotodetector. Este método limita las medidas en fluidos transparentes, la portabilidad del instrumento, e incrementa la complejidad de medición multiplexada. El desarrollo de cantilevers sensibles a la deflexión mediante la integración de piezoresistores o transistores de efecto de campo (MOSFET) implementados en el mismo voladizo, resuelve este problema. Sin embargo, simultáneamente se disminuye la resolución del sensor debido al incremento del ruido electrónico. Por otro lado, se puede detectar moléculas midiendo la fuerza de enlace entre una molécula y su receptor, estirando el complejo molecular, mediante espectroscopia de fuerza atómica (AFS), técnica basada en el microscopio de fuerza atómica (AFM). A pesar de la elevada resolución en fuerza, el AFM no ha logrado aún convertirse en instrumento analítico debido principalmente a la complejidad del mismo y de su uso. Un biosensor basado en cantilevers que puedan detectar su propia deflexión y que emplee la AFS, tendría resolución de una molécula, podría ser utilizado en fluidos opacos, tendría potencial de multiplexado y su integración a una celda microfluídica sería viable. Considerando esto, se desarrollaron cantilevers dotados de resolución de pN y compatibles con líquidos. Se diseñaron y modelaron cantilevers basados en silicio cristalino y se ha optimizado el proceso de fabricación para aumentar la sensibilidad y el rendimiento. Asímismo, se ha trabajado sobre el modelo, el desarrollo y la fabricación de cantilevers con un MOSFET integrado. Se concluye que el primer sensor ofrece una solución tecnológica más directa, aunque el segundo puede ser una buena alternativa. Simultáneo a la fabricación de sensores, se desarrollaron también nuevas técnicas y montajes para la rápida caracterización eléctrica y electromecánica de los sensores de manera precisa y fiable. Esto fue crucial a la hora de validar el proceso de producción y los dispositivos finales. Después de obtener muy alta resolución (<10 pN en líquido) con elevado rendimiento en la producción, los sensores fueron utilizados para el estudio de procesos de reconocimiento molecular entre avidina y biotina. Para lograr este objetivo, los sensores fueron integrados en un AFM comercial para aprovechar su elevada estabilidad mecánica y el desplazamiento nanométrico del piezoactuador. Se detectaron con éxito las fuerzas de enlace relacionadas a la formación del complejo molecular biotina-avidina, resaltando de esta manera, la posibilidad de detección label-free de biomoléculas en condiciones cuasi fisiológicas con resolución de una molécula. Además de la elevada sensibilidad, estos sensores pueden utilizarse sin restricciones en fluidos opacos, se pueden integrar fácilmente en celdas microfluídicas y demuestran capacidad para el multiplexado. Este resultado abre nuevas perspectivas en detectores de marcadores biológicos con elevada sensibilidad y que puedan trabajar en condiciones fisiológicas.Biorecognition processes between receptors and their conjugate ligands are very important in biology. These biomolecules can build up very specific complexes displaying a variety of functions such as genome replication and transcription, enzymatic activity, immune response, cellular signaling, etc. The unambiguous one-to-one complementarity exhibited by these biological partners is widely exploited also in biotechnology to develop biosensors. Depending on the nature of the transduction signals, biosensors can be classified in optical, electrical and mechanical. Among mechanical biosensors, the microcantilevers play a prominent role. They have been used as stress or mass transducers in biomolecules detection for already more than a decade. The binding of molecules to their functionalized surface is detected by measuring either the deflection in static mode or the resonant frequency shift in dynamic mode. The deflection of the cantilever is converted optically by a laser and a photodetector in order to have the highest possible resolution. This limits the measurements in transparent liquids, the portability of the instrument and increases the complexity for multiplexing. The development of self-sensing cantilevers by integrating piezoresistors or metal-oxide-semiconductor field effect transistors (MOSFET) into the cantilever solves this issue. However, at the same time, this decreases the bending and frequency shift resolution due to the higher transducer noise. On the other hand, the detection of a single molecule can be attained measuring the unbinding force between two molecules of a complex pulling them apart, using the atomic force spectroscopy (AFS) measuring approach. This technique is based on the atomic force microscope (AFM). Despite the high force resolution, AFM has still not become an analytical instrument and it is mainly due to the complexity of the instrument and of its use. A biosensor based on AFS and on self-sensing cantilever would allow single molecule resolution, working in opaque fluids, easy multiplexing capability, and relatively easy integration in microfluidics cells. In this perspective, we worked to obtain self sensing-probes endowed with pN resolution and compatible with liquid media. Cantilevers based on single crystalline silicon have been modeled and the fabrication process has been optimized to improve the force sensitivity and to obtain high fabrication yield. At the same time we worked also on the modeling, development and fabrication of cantilevers with embedded MOSFET piezoresistive transducers. It turned out that the probes with integrated piezoresistor offer a more straightforward solution, but also the MOSFET cantilever can offer a good alternative. Alongside the force sensors fabrication, new high-throughput set-ups and techniques have been developed and optimized to measure the electrical and electromechanical characteristics of micro-electro-mechanical systems (MEMS) in a precise and reliable way. This was of key importance to correctly validate the new technological processes involved in production as well as characterize the final devices. After achieving very good sensor performances (resolution < 10 pN in liquid environment) with high production yield, we used the force probes to investigate the biorecognition processes in the avidin-biotin complex. For this purpose we integrated the sensor into a commercial AFM to take advantage of the high mechanical stability of this equipment and the highly reliable displacement of the piezo actuator. We detected the forces related to the avidin-biotin complex formation, highlighting the possibility of biomolecule label-free recognition in nearly physiological conditions and at single molecule resolution. Beside the very high sensitivity attained, the sensor can be used with no restrictions in opaque media; it can be easily integrated in microfluidic cells and it displays a high multiplexing potentiality. This result opens new perspectives in highly sensitive label free biomarkers detectors in nearly physiological conditions

Silicon microcantilevers with MOSFET detection

We report the fabrication of silicon microcantilevers with MOSFET detection, to be used in force measurements for biomolecular detection. Thin cantilevers are required for a high force sensitivity. Therefore the source and drain of the transistors have been fabricated by As implantation to obtain shallow PN junctions. The cantilevers have been oriented on the non-standard (100) crystallographic direction of silicon, to maximize the stress response of the NMOS transistors. The force sensitivity and resolution of the cantilevers have been tested by applying a force with an AFM tip. Values of 25 μV/pN and 56 pN respectively have been obtained for a force applied at the tip of a cantilever with a length of 200 μm, a width of 24 μm and a silicon thickness of 340 nm.Plan Nacional MICINN TEC2007-65692; NANOSELECT–CSD2007-00041 Consolider-Ingenio 2010.Peer reviewe

Silicon microcantilevers with MOSFET detection

We report the fabrication of silicon microcantilevers with MOSFET detection, to be used in force measurements for biomolecular detection. Thin cantilevers are required for a high force sensitivity. Therefore the source and drain of the transistors have been fabricated by As implantation to obtain shallow PN junctions. The cantilevers have been oriented on the non-standard (1 0 0) crystallographic direction of silicon, to maximize the stress response of the NMOS transistors. The force sensitivity and resolution of the cantilevers have been tested by applying a force with an AFM tip. Values of 25 μV/pN and 56 pN respectively have been obtained for a force applied at the tip of a cantilever with a length of 200 μm, a width of 24 μm and a silicon thickness of 340 nm

Force sensors based on piezoresistive and MOSFET cantilevers for biomolecular sensing

Los procesos de reconocimiento biomolecular entre receptores y ligandos son muy importantes en biología. Estas biomoléculas pueden desarrollar complejos muy específicos y tener una variedad de funciones como replicación y transcripción genómica, actividad enzimática, respuesta inmune, señalamiento celular, etc. La complementariedad inequívoca mostrada por estos componentes biológicos es ampliamente utilizada para desarrollar biosensores. Dependiendo de la naturaleza de las señales que se convierten, los biosensores pueden ser clasificados en ópticos, eléctricos o mecánicos. Entre los sensores mecánicos, los microcantilevers son los más comunes. Han sido utilizados como sensores de estrés superficial o como sensores de masa en detección de biomoléculas, desde hace más de 10 años. El enlace de las moléculas a sus superficies funcionalizadas se puede detectar midiendo la deflexión en modo estático o la variación de la frecuencia de resonancia en modo dinámico. Para lograr la máxima resolución, la deflexión es medida por un láser y un fotodetector. Este método limita las medidas en fluidos transparentes, la portabilidad del instrumento, e incrementa la complejidad de medición multiplexada. El desarrollo de cantilevers sensibles a la deflexión mediante la integración de piezoresistores o transistores de efecto de campo (MOSFET) implementados en el mismo voladizo, resuelve este problema. Sin embargo, simultáneamente se disminuye la resolución del sensor debido al incremento del ruido electrónico. Por otro lado, se puede detectar moléculas midiendo la fuerza de enlace entre una molécula y su receptor, estirando el complejo molecular, mediante espectroscopia de fuerza atómica (AFS), técnica basada en el microscopio de fuerza atómica (AFM). A pesar de la elevada resolución en fuerza, el AFM no ha logrado aún convertirse en instrumento analítico debido principalmente a la complejidad del mismo y de su uso. Un biosensor basado en cantilevers que puedan detectar su propia deflexión y que emplee la AFS, tendría resolución de una molécula, podría ser utilizado en fluidos opacos, tendría potencial de multiplexado y su integración a una celda microfluídica sería viable. Considerando esto, se desarrollaron cantilevers dotados de resolución de pN y compatibles con líquidos. Se diseñaron y modelaron cantilevers basados en silicio cristalino y se ha optimizado el proceso de fabricación para aumentar la sensibilidad y el rendimiento. Asímismo, se ha trabajado sobre el modelo, el desarrollo y la fabricación de cantilevers con un MOSFET integrado. Se concluye que el primer sensor ofrece una solución tecnológica más directa, aunque el segundo puede ser una buena alternativa. Simultáneo a la fabricación de sensores, se desarrollaron también nuevas técnicas y montajes para la rápida caracterización eléctrica y electromecánica de los sensores de manera precisa y fiable. Esto fue crucial a la hora de validar el proceso de producción y los dispositivos finales. Después de obtener muy alta resolución ( 10 pN en líquido) con elevado rendimiento en la producción, los sensores fueron utilizados para el estudio de procesos de reconocimiento molecular entre avidina y biotina. Para lograr este objetivo, los sensores fueron integrados en un AFM comercial para aprovechar su elevada estabilidad mecánica y el desplazamiento nanométrico del piezoactuador. Se detectaron con éxito las fuerzas de enlace relacionadas a la formación del complejo molecular biotina-avidina, resaltando de esta manera, la posibilidad de detección label-free de biomoléculas en condiciones cuasi fisiológicas con resolución de una molécula. Además de la elevada sensibilidad, estos sensores pueden utilizarse sin restricciones en fluidos opacos, se pueden integrar fácilmente en celdas microfluídicas y demuestran capacidad para el multiplexado. Este resultado abre nuevas perspectivas en detectores de marcadores biológicos con elevada sensibilidad y que puedan trabajar en condiciones fisiológicas.Biorecognition processes between receptors and their conjugate ligands are very important in biology. These biomolecules can build up very specific complexes displaying a variety of functions such as genome replication and transcription, enzymatic activity, immune response, cellular signaling, etc. The unambiguous one-to-one complementarity exhibited by these biological partners is widely exploited also in biotechnology to develop biosensors. Depending on the nature of the transduction signals, biosensors can be classified in optical, electrical and mechanical. Among mechanical biosensors, the microcantilevers play a prominent role. They have been used as stress or mass transducers in biomolecules detection for already more than a decade. The binding of molecules to their functionalized surface is detected by measuring either the deflection in static mode or the resonant frequency shift in dynamic mode. The deflection of the cantilever is converted optically by a laser and a photodetector in order to have the highest possible resolution. This limits the measurements in transparent liquids, the portability of the instrument and increases the complexity for multiplexing. The development of self-sensing cantilevers by integrating piezoresistors or metal-oxide-semiconductor field effect transistors (MOSFET) into the cantilever solves this issue. However, at the same time, this decreases the bending and frequency shift resolution due to the higher transducer noise. On the other hand, the detection of a single molecule can be attained measuring the unbinding force between two molecules of a complex pulling them apart, using the atomic force spectroscopy (AFS) measuring approach. This technique is based on the atomic force microscope (AFM). Despite the high force resolution, AFM has still not become an analytical instrument and it is mainly due to the complexity of the instrument and of its use. A biosensor based on AFS and on self-sensing cantilever would allow single molecule resolution, working in opaque fluids, easy multiplexing capability, and relatively easy integration in microfluidics cells. In this perspective, we worked to obtain self sensing-probes endowed with pN resolution and compatible with liquid media. Cantilevers based on single crystalline silicon have been modeled and the fabrication process has been optimized to improve the force sensitivity and to obtain high fabrication yield. At the same time we worked also on the modeling, development and fabrication of cantilevers with embedded MOSFET piezoresistive transducers. It turned out that the probes with integrated piezoresistor offer a more straightforward solution, but also the MOSFET cantilever can offer a good alternative. Alongside the force sensors fabrication, new high-throughput set-ups and techniques have been developed and optimized to measure the electrical and electromechanical characteristics of micro-electro-mechanical systems (MEMS) in a precise and reliable way. This was of key importance to correctly validate the new technological processes involved in production as well as characterize the final devices. After achieving very good sensor performances (resolution 10 pN in liquid environment) with high production yield, we used the force probes to investigate the biorecognition processes in the avidin-biotin complex. For this purpose we integrated the sensor into a commercial AFM to take advantage of the high mechanical stability of this equipment and the highly reliable displacement of the piezo actuator. We detected the forces related to the avidin-biotin complex formation, highlighting the possibility of biomolecule label-free recognition in nearly physiological conditions and at single molecule resolution. Beside the very high sensitivity attained, the sensor can be used with no restrictions in opaque media; it can be easily integrated in microfluidic cells and it displays a high multiplexing potentiality. This result opens new perspectives in highly sensitive label free biomarkers detectors in nearly physiological conditions

Magnetic properties of cobalt microwires measured by piezoresistive cantilever magnetometry

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. (CC BY-NC-ND 3.0).We present the magnetic characterization of cobalt wires grown by focused electron beam-induced deposition (FEBID) and studied using static piezoresistive cantilever magnetometry. We have used previously developed high force sensitive submicron-thick silicon piezoresistive cantilevers. High quality polycrystalline cobalt microwires have been grown by FEBID onto the free end of the cantilevers using dual beam equipment. In the presence of an external magnetic field, the magnetic cobalt wires become magnetized, which leads to the magnetic field dependent static deflection of the cantilevers. We show that the piezoresistive signal from the cantilevers, corresponding to a maximum force of about 1 nN, can be measured as a function of the applied magnetic field with a good signal to noise ratio at room temperature. The results highlight the flexibility of the FEBID technique for the growth of magnetic structures on specific substrates, in this case piezoresistive cantileversThis work was supported by the Spanish Ministry of Economy and Competitiveness through projects No. TEC2011-23600, Nanoselect-CSD2007-00041 (Consolider-Ingenio 2010 programme) and MAT2011- 27553-C02, including FEDER funds, by the Aragón Regional Government, and by the EU Sudoe Interreg TRAIN2 proyect.Peer Reviewe

Cyclin T2A gene maps on human chromosome 2q21.

Cyclin T2a was recently identified as one of the regulatory subunits of the cdk-cyclin complex P-TEFb, the most studied positive factor in the regulation of transcription elongation. By fluorescent in situ hybridization (FISH), the gene codifying for cyclin T2a has been mapped on human chromosome 2q21. This locus also has been linked to different forms of myopathy. By use of a new specific antiserum raised against cyclin T2a, the immunohistochemical pattern of expression of cyclin T2a in human tissues has been examined and compared to that of cyclin T1, described in the previous report. The observation that immunohistochemical expression of cyclin T2a was high in skeletal muscle cells, whereas it was undetectable in two cases of centronuclear myopathy, together with its chromosomal location, suggests an involvement of the cdk9-cyclin T2a complex in this disease

Cyclin T2a gene maps on human chromosome 2q21

Cyclin T2a was recently identified as one of the regulatory subunits of the cdk-cyclin complex P-TEFb, the most studied positive factor in the regulation of transcription elongation. By fluorescent in situ hybridization (FISH), the gene codifying for cyclin T2a has been mapped on human chromosome 2q21. This locus also has been linked to different forms of myopathy. By use of a new specific antiserum raised against cyclin T2a, the immunohistochemical pattern of expression of cyclin T2a in human tissues has been examined and compared to that of cyclin T1, described in the previous report. The observation that immunohistochemical expression of cyclin T2a was high in skeletal muscle cells, whereas it was undetectable in two cases of centronuclear myopathy, together with its chromosomal location, suggests an involvement of the cdk9-cyclin T2a complex in this disease