DOCTORAL THESIS
Development of a biosensor based on
a
-SixC1-x:H to detect enterotoxigenic
Escherichia coli
Submitted by
M. Sc. José Luis Herrera Celis
in partial fulfillment of the requirements
for the degree
Doctor of Science in Electronics
In the Department of Electronics at the
National Institute for Astrophysics,
Optics and Electronics
Advisor: Dr. Claudia Reyes Betanzo
Co-advisor: Dr. Abdu Orduña Díaz
Tonantzintla, November 2016
©INAOE 2016
Copyright
The author gives the INAOE permission to
reproduce and distribute copies of this thesis in
whole or in parts.
I
ACKNOWLEDGEMENTS
It is my wish to offer these lines to the people and institutions that contributed to the
development and successful conclusion of this PhD thesis. Firstly, I am pleased with the help,
support and comprehension from my advisors Dr. Claudia Reyes Betanzo and Dr. Abdu
Orduña Díaz during the four years of my studies. They were always attentive to resolve any
issues related to the equipment and materials needed for each manufacturing process and
each experiment.
I would like to thank the technicians of the Laboratorio de Microelectrónica of the Instituto Nacional de Astrofísica, Óptica y Electrónica (INAOE) Adrián Itzmoyotl Toxqui, Armando Hernández Flores, Víctor Aca Aca, José E. Pablo Alarcón Peña and Ignacio Juárez Ramírez
for their support and assistance during the manufacture and measurement of the interdigitated
microelectrode arrays. Their knowledge and expertise contributed to the progress of the
activities according to the schedule.
In a special way I want to thank Dr. Daniela Díaz Alonso for her support and teaching about
the CoventorWare® software. I would also like to thank Dr. Carlos Ramírez Nezahualcoyotl and Leticia Tecuapetla Quechol of the Laboratorio de Microscopía Electrónica of the
INAOE who collaborated me with the measurements of SEM, EDS and AFM, and Orlando Zaca Moran of the Centro de Investigación en Biotecnología Aplicada (CIBA) for their collaboration during FTIR measurements.
As for the biofunctionalization process of surfaces, I would like to express my thanks to Ana
Laura Pérez Coyotl who, as part of her thesis of master, tuned the parameters of the process
for a-SixC1-x:H surfaces. During the development of the processes, I was also accompanied by Janet Morales Chávez and Arely Culebro Gomez who collaborated and assisted me with
their comments and suggestions, therefore I am grateful to them.
The cytotoxicity assessment was carried out at the University of South Florida (USF) under the supervision of Dr. Stephen E. Saddow and with the collaboration of Katie Noble, Evans
II
for the kind treatment I received during the two months. In addition to this, I would like to
thank Dr. Stephen E. Saddow for approving my stay at USF, for his collaboration with the legal procedures, for his continued concern for my well-being and because we had the
materials and the tools required for the tests, and above all for his teachings.
Two important elements of this work have been the bacteria of enterotoxigenic Escherichia coli (E. coli) and the polyclonal anti-E. coli antibodies. Regarding to the bacteria, I am thankful to Dr. Elsa Iracena Castañeda Roldán of the Laboratorio de Patogenicidad Microbiana of the Departamento de Investigaciones Microbiológicas in the Instituto de Ciencias of the Benemérita Universidad Autónoma de Puebla (ICUAP) for the supply of the strain of the bacteria, and for his cooperation and support with the aspects related with this
bacterium. And as for the antibodies, I am thankful to Zeus Saldaña Ahuactz of the
Laboratorio de Investigación en Bacteriología Intestinal of the Hospital Infantil de México Federico Gómez (HIMFG)who managed the donation of the antibodies for this research.
I do not want to miss this opportunity to thank all the researchers of the microelectronics area
of the INAOE for their attention and contributions to this work, as well as for the time they devoted to my human and professional training. I also thank Dr. Alfonso Torres Jacome, Dr.
Mario Moreno Moreno and Dr. Guillermo Espinosa Flores-Verdad of the INAOE, and Dr. Marlon Rojas López and Dr. José Francisco Sánchez Ramírez of the CIBA, who improved this thesis acting as thesis jury members. Especially I am grateful to Dr. Alfonso Torres
Jacome for listening to me when there were difficulties, as well as for his teachings on
deposition of amorphous materials by PECVD and manufacturing processes of sensors.
There are four institutions that contributed significantly to my doctoral studies and whose
participation is worth mentioning. I am grateful to INAOE for allowing me to carry out both my master and doctoral studies. INAOE is tattooed in my heart. I thank CIBA for borrowing me its installations to do the experiments related to the biofunctionalization process.
Similarly, I thank USF for making possible the stay during which the cytotoxicity tests were performed. Finally, I appreciate the doctoral scholarship as well as the support under the
project No. 242440 provided by Consejo Nacional de Ciencia y Tecnología (CONACYT) because thanks to these economic supports my research could come to fruition.
III
DEDICATION
To my parents…
…who strove to give me education and much more,
to my siblings…
…who always trusted me and cleared the way for me,
to my nieces and nephews…
…who have been my main motivation,
to my girlfriend Emilia Margarita Méndez Aguilar…
…who has been by my side in difficult times,
and to my friends Diana González, Susana Romero, Leticia Tecuapetla, Martha Matiz, Olga Roa, Jessenia Chirinos, Adriana Tepaneca, Carolina Acuña, Dulce Murias, José de Jesús Martínez, Fabián Díaz, Alexander Gómez, Mario Betancurt, Cristian Martínez, Arcesio Arbeláez, Adrián Itzmoyotl, Iván Carvajal, Sergio Fuentes, Armando Hernández, Carlos Ospina, Harold Peña and Fabián Núñez…
…who in one way or another, being far or near, have always had words of encouragement
IV
ABSTRACT
The food industry and clinical analysis, among other sectors, require the development of
techniques and devices that detect pathogens, while the development of implantable devices
needs biocompatible materials with low degradation in biological environment to increase
the lifetime of the device. Throughout this work, hydrogenated amorphous silicon-carbon
(a-SixC1-x:H) alloy is proposed, obtained, characterized and incorporated into the development of a proposed interdigitated microelectrode array (PIMA) to capture the bacteria
of enterotoxigenic Escherichiacoli (E. coli, ETEC). a-SixC1-x:H is obtained by the technique of plasma-enhanced chemical vapor deposition (PECVD) using methane and silane as precursor gases under high hydrogen dilution and low power density in order to improve its
biocompatibility. Furthermore, considering the projection of the material in biosensors and
the effect of the precursor gas ratio on the material properties, films with different precursor
gas ratio were deposited and characterized. The result was a set of films with low density of
CHn groups, which was also subject under studied.
Simultaneously, the PIMA was designed and simulated using CoventorWare® software.
Structurally the PIMA incorporates two layers of a-SixC1-x:H, one between and another on microelectrode, on which a biofunctionalization layer is formed. The aim of this layer is to
capture the bacteria by affinity interactions. Functionally the PIMA is a transducer based on
electrical impedance, namely the capture of E. coli bacteria causes changes in the electrical properties of the medium between and on the microelectrodes of the array, which are
associated with changes in electrical impedance. The simulations were made with the purpose
of knowing the operation that the PIMA would have under operating conditions (with
bacterial environment) and of analyzing the design aspects that could affect or increase the
sensitivity of the array. One of the results of the simulations was that the conductivity of the
layer of a-SixC1-x:H on microelectrodes should be as high as possible to reduce its effect on the sensitivity of the biosensor, therefore diborane as doping gas was incorporated during the
V
Once selected the deposition parameters under which a high density of Si–C bonds is
obtained in the films and made the respective depositions, cytotoxicity assessments were
carried out on five films. And as part of the development of the PIMA, a biofunctionalization
process for a-SixC1-x:H surfaces was designed and implemented. The results of the cytotoxicity assessments revealed the effect of the incorporation of CHn groups on the
cytotoxicity of the films, while the spectra of FTIR spectroscopy measurements as technique for monitoring the process showed that bacteria were captured by polyclonal anti-E. coli
antibodies present in the surface of the biofunctionalization layer. These results enabled
advancing the development of the PIMA.
The fabrication of the PIMA included eight stages, within which are two stages of
photolithography, the wet etching of titanium and the plasma-based etching of a-SixC1-x:H. In this regard, the fact of having a layer of doped a-SixC1-x:H on top of microelectrodes had advantages and disadvantages in terms of fabrication and operation. From the fabrication
standpoint, it masks the titanium during the corresponding wet etching. In terms of operation,
this layer enables to capture bacteria on microelectrodes, increasing the sensing area and
therefore the sensitivity of the biosensor, but also gives rise to a space charge region at the
interface with titanium. It appears in the impedance spectrum of the biosensor as a contact
resistance and a capacitance related to the space charge region.
Finally, the PIMA is bio-functionalized and both previous stages of biofunctionalization and
the capture event of E. coli bacteria is recorded by changes in the impedance spectrum of the biosensor using electrical impedance spectroscopy measurements in the range from 1 kHz to
5 MHz. The percentage change in the magnitude of the electrical impedance reached
133.37% (impedance response of 1.51 MΩ) at 28.5 kHz when 30 µL of bacteria in
Luria-Bertani medium (LB) at a concentration of 107 CFU/ml were put on the array for 25 minutes.
VI
RESUMEN
La industria alimenticia y el análisis clínico, entre otros sectores, requieren el desarrollo de
técnicas y dispositivos que detecten organismos patógenos, mientras que el desarrollo de
dispositivos implantables necesita materiales biocompatibles con baja degradación en medio
biológico para aumentar el tiempo de vida del dispositivo. A lo largo de este trabajo, una
aleación de silicio-carbono amorfo hidrogenado (a-SixC1-x:H) es propuesto, obtenido, caracterizado e incorporado en el desarrollo de un arreglo de microelectrodos interdigitados
propuesto (PIMA por sus siglas en inglés) para capturar la bacteria de Escherichia coli
enterotoxigénica (E. coli, ETEC por sus siglas en inglés). a-SixC1-x:H es obtenido por la técnica de depósito en fase vapor asistido por plasma (PECVD por sus siglas en inglés) usando metano y silano como gases precursores bajo condiciones de alta dilución en
hidrógeno y baja densidad de potencia con el propósito de mejorar su biocompatibilidad.
Además, teniendo en cuenta la proyección del material en biosensores y la influencia de la
razón de gases precursores sobre las propiedades del material, películas con diferentes
razones de gases precursores fueron depositadas y caracterizadas. El resultado fue un
conjunto de películas con baja densidad de grupos CHn, lo cual fue también objeto de estudio.
En simultáneo, el PIMA fue diseñado y simulado usando el software CoventorWare®. Estructuralmente el PIMA incorpora dos capas de a-SixC1-x:H, una entre microelectrodos y otra sobre microelectrodos, sobre las cuales una capa de biofuncionalización es formada. Esta
capa tiene como objetivo capturar la bacteria por medio de interacciones de afinidad.
Funcionalmente el PIMA es un transductor basado en impedancia eléctrica, es decir, la
captura de bacterias de E. coli genera cambios en las propiedades eléctricas del medio entre y sobre los microelectrodos del arreglo, los cuales son asociados a los cambios en la
impedancia eléctrica. Las simulaciones fueron hechas con el propósito de conocer el
funcionamiento que tendría el PIMA bajo condiciones de operación (con medio bacterial) y
de analizar aspectos de diseño que podrían afectar o incrementar la sensitividad del arreglo.
VII
sobre microelectrodos debería ser tan alta como fuera posible para reducir su efecto sobre la
sensitividad del biosensor, por lo tanto, diborano como gas dopante fue incorporado en los
depósitos de algunas películas, las cuales también fueron caracterizadas.
Una vez seleccionados los parámetros de depósito bajo los cuales una alta densidad de
enlaces Si–C es obtenida en las películas y efectuados los respectivos depósitos, pruebas de
citotoxicidad fueron realizadas sobre cinco películas. Y como parte del desarrollo del PIMA,
un proceso de biofuncionalización para superficies de a-SixC1-x:H fue diseñado y aplicado. Los resultados de las pruebas de citotoxicidad revelaron la incidencia de la incorporación de
grupos CHn en la citotoxicidad de las películas, mientras que los espectros de las mediciones
de espectroscopia FTIR como técnica de monitoreo del proceso mostraron que las bacterias eran capturadas por los anticuerpos policlonales anti-E. coli presentes en la superficie de la capa de biofuncionalización. Estos resultados permitieron avanzar en el desarrollo del PIMA.
La fabricación del PIMA incluyó ocho etapas, dentro de las cuales figuran dos etapas de
fotolitografía, el grabado húmedo de titanio y el grabado por plasma de a-SixC1-x:H. Al respecto, el hecho de contar con una capa de a-SixC1-x:H dopado sobre microelectrodos tuvo ventajas y desventajas en términos de fabricación y funcionamiento. Desde el punto de vista
de fabricación, ésta enmascara el titanio durante el correspondiente grabado húmedo. En
cuanto a funcionamiento, esta capa posibilita capturar bacterias sobre microelectrodos,
aumentando el área de sensado y por ende la sensitividad del biosensor, pero a la vez da
origen a una región de carga espacial en la interfaz con titanio. Esto aparece en el espectro
de impedancia del biosensor como una resistencia de contacto y una capacitancia asociada a
la región de carga espacial.
Finalmente, el PIMA es biofuncionalizado y tanto las etapas previas de biofuncionalización
como el evento de captura de las bacterias de E. coli es registrado por variaciones en el espectro de impedancia del biosensor utilizando mediciones de espectroscopia de impedancia
eléctrica en el rango de 1 kHz a 5 MHz. El cambio porcentual en la magnitud de la
impedancia eléctrica alcanzó el 133.37% (respuesta en impedancia de 1.51 MΩ) at 28.5 kHz
cuando 30 µL de bacterias en medio Luria-Bertani a una concentración de 107 CFU/ml fueron puestos sobre el arreglo durante 25 minutos.
VIII
CONTENTS
CHAPTER 1
Hydrogenated amorphous silicon-carbon alloy (
a
-Si
xC
1-x:H) and its
prospects in biosensors
1. Introduction ... 1
2. Deposition of a-SixC1-x:H by PECVD ... 1
3. a-SixC1-x:H structure ... 3
4. Biosensors ... 7
5. Applications of a-SixC1-x:H in biosensors ... 8
6. Immunosensors... 10
7. Electrical impedance spectroscopy ... 13
References ... 16
CHAPTER 2
Design and simulation of the proposed interdigitated microelectrode array
(PIMA) to detect
E. coli
1. Introduction ... 212. Equivalent circuit and design parameters of an IMA ... 21
3. Proposed interdigitated microelectrode array (PIMA) ... 26
4. Electrical model of the PIMA in solution with E. coli ... 28
5. Design and simulation of the PIMA to detect E. coli... 32
5.1. Doping level of the a-SixC1-x:H layer on top of microelectrodes ... 35
5.2. Influence of the entrapping site of E. coli ... 35
IX
5.4. Sensitivity analysis ... 38
6. Required properties of a-SixC1-x:H layers to be applied to the PIMA ... 40
7. Conclusions ... 41
References ... 42
CHAPTER 3
Deposition processes and characterization of intrinsic and B-doped
a
-Si
xC
1-x:H films
1. Introduction ... 442. Effect of the hydrogen dilution during a-SixC1-x:H deposition ... 44
3. Deposition processes of a-SixC1-x:H films by PECVD at 110 kHz ... 46
4. Deposition processes of a-SixC1-x:H by PECVD at 13.56 MHz ... 48
5. Morphological, chemical, optical and electrical characterization ... 49
6. Properties of a-SixC1-x:H films deposited at 110 kHz ... 54
7. Properties of a-SixC1-x:H films deposited at 13.56 MHz ... 61
8. Deposition processes and characterization of B-doped a-SixC1-x:H films ... 70
9. Conclusions ... 75
References ... 75
CHAPTER 4
Cytotoxicity assessment and biofunctionalization process of
a
-Si
xC
1-x:H
films
1. Introduction ... 772. Cytotoxicity assessment: the extract method ... 77
2.1. Selection, cleaning and sterilization of materials ... 78
2.2. Cell culture ... 79
2.3. Production of extracts and cell culture ... 81
X
2.5. Quantification of cytotoxicity ... 84
3. Results of the cytotoxicity assessment ... 85
4. Biofunctionalization process of a-SixC1-x:H surfaces to capture E. coli ... 90
4.1. Hydroxylation... 92
4.2. Silanization ... 92
4.3. Generation of aldehyde groups ... 93
4.4. Binding via PrA ... 94
4.5. Immobilization of anti-E. coli polyclonal antibodies ... 95
4.6. Entrapping of enterotoxigenic Escherichia coli in Luria-Bertani medium ... 95
5. Monitoring of the biofunctionalization process by FTIR measurements ... 97
6. Conclusions ... 100
References ... 100
CHAPTER 5
Fabrication of the proposed interdigitated microelectrode array (PIMA) to
detect
E. coli
1. Introduction ... 1032. Structure of the proposed interdigitated microelectrode array (PIMA) ... 103
3. Stages of the fabrication process of the proposed interdigitated microelectrode array (PIMA) ... 106
3.1. Design and manufacture of masks ... 107
3.2. Photolithography (Level 1 mask) ... 109
3.3. B-doped a-SixC1-x:H etching (array) ... 111
3.4. Ti etching (array) ... 112
3.5. Photoresist removal ... 115
3.6. Photolithography (Level 2 mask) ... 115
3.7. B-doped a-SixC1-x:H etching (pads) ... 116
3.8. Photoresist removal ... 117
XI
5. Measuring of PIMAs by electrical impedance spectroscopy ... 120
6. Conclusions ... 125
References ... 126
CHAPTER 6
Analysis of suitability of
a
-Si
xC
1-x:H films for biomedical applications and
biosensors
1. Introduction ... 1272. Analysis of the results of the cytotoxicity assessments... 127
3. Analysis of the electrical measurements of the PIMAs ... 132
4. Biofunctionalization test of the PIMA to detect E. coli ... 136
5. Conclusions ... 138
6. Derivative works ... 140
6.1. Publications in journals ... 140
6.2. Proceedings ... 140
6.3. International congresses ... 141
6.4. National congresses ... 142
7. Future work ... 143
XII
LIST OF FIGURES
Fig. 1.PECVD reactor coupled a) inductively and b) capacitively [3]. ... 2
Fig. 2. Atomic structures of carbon as a) graphite and b) diamond. ... 4
Fig. 3. Atomic structures of silicon carbide. a) Diamond-like and b) graphite-like structures. ... 4
Fig. 4. Atomic structures of hydrogenated amorphous silicon-carbon alloy. ... 5
Fig. 5. FTIR spectrum of hydrogenated amorphous silicon-carbon alloy. ... 6
Fig. 6. Schematic diagram of the inmunoglobulin G (IgG) antibody [44]. ... 11
Fig. 7. Specific interaction of an antibody with a mixture of antigens resulting in an antibody-antigen complex [44]. ... 11
Fig. 8. Faradaic process model. a) Equivalent electrical circuit and b) Nyquist diagram of the equivalent circuit [45]. ... 15
Fig. 9. Non-faradaic process models [47]. ... 15
Fig. 10. Layout of an interdigitated microelectrode array with a width w and length p of the fingers, and a spacing s between them. ... 22
Fig. 11. Energy levels in a metal electrode (left-hand side) and an electrolyte (right-hand side) with a common vacuum reference scale. ϕ is the work function of the metal [7]. ... 23
Fig. 12. Schematic of the charge distribution at the electrode/electrolyte and built-in potential distribution through the double dielectric layer (Stern model). IHP is the inner Helmholtz plane and OHP is the outer Helmholtz plane. ... 23
Fig. 13. Equivalent circuit of an IMA in solution represented by the double layer capacitance (CDL), the resistance of the solution (RSol) and the dielectric capacitance (Cdi). ... 25
Fig. 14. Diagram of the impedance of an IMA against the frequency. The values of the low and high cutoff frequencies (Flow and Fhigh, respectively) depend on the capacitance of the double dielectric layer (CDL), the resistance of the solution RSol and the capacitance of the array (Cdi) due to the permittivity of the solution [3]. ... 26
Fig. 15. Structure of the proposed interdigitated microelectrode array (PIMA) versus structure of the conventional interdigitated microelectrode array (CIMA). a) Top view of the CIMA, b) cross-section of the CIMA, c) top view of the PIMA and d) cross-section of the PIMA. ... 27
Fig. 16. Equivalent circuit of the PIMA in solution without analyte. ... 28
Fig. 17. Bacterium of Escherichia coli (E. coli) and its main parts [11]. ... 29
Fig. 18. Equivalent circuit of the PIMA in solution with bacteria both between and on microelectrodes. The subscript MOE and COE mean membrane on electrodes and cytoplasm on electrodes, respectively, while the subscript MBE and CBE mean membrane between electrodes and cytoplasm between electrodes, respectively. ... 30
XIII
Fig. 19. Simplified equivalent circuit of the PIMA in solution with bacteria both between and on
microelectrodes. ... 31
Fig. 20. Cross-section of a pair of microelectrodes of the array designed for simulations in CoventorWare® software with bacteria between and on top of them. ... 34
Fig. 21. Simulated electrical impedance of the PIMA with conductivities of the a-SixC1-x:H thin film on top of microelectrodes from 10-8 S/cm (undoped) to 10-3 S/cm (doped). ... 35
Fig. 22. Simulation schemes of the PIMA with the doped a-SixC1-x:H layer on top of microelectrodes. a) PIMA in neutral phosphate buffer (NPB) without bacteria, b) PIMA in NPB with bacteria between microelectrodes, c) PIMA in NPB with bacteria on half the sensing area, d) PIMA in NPB with bacteria on microelectrodes and e) PIMA in NPB with bacteria on the total sensing area. ... 36
Fig. 23. Simulated response of the PIMA with the a-SixC1-x:H layer on microelectrodes doped (conductivity of 10-3 S/cm). a) PIMA in neutral phosphate buffer (NPB) without bacteria, b) PIMA in NPB with bacteria between microelectrodes, c) PIMA in NPB with bacteria on half the sensing area, d) PIMA in NPB with bacteria on microelectrodes and e) PIMA in NPB with bacteria on the total sensing area. ... 37
Fig. 24. Operation regions of the proposed interdigitated microelectrode array (PIMA). ... 38
Fig. 25. Simulation schemes of the PIMA and the CIMA with and without bacteria covering the whole sensing area of each one. a) CIMA in neutral phosphate buffer (NPB) without bacteria, b) CIMA in NPB with bacteria between microelectrodes, c) PIMA in NPB without bacteria and d) PIMA in NPB without bacteria between and on top of the microelectrodes. ... 39
Fig. 26. Impedance spectra of the CIMA and the PIMA without and with E. coli on the maximum sensing area. ... 39
Fig. 27. Maximum percentage change (MPC) of the impedance spectra of the CIMA and the PIMA under bacterial detection. ... 40
Fig. 28. Illustration of the density-of-states (DOS), together with the electron distribution nBT(E). ... 45
Fig. 29.PECVD reactor, AMP PLASMA II 3300 model. ... 47
Fig. 30. PECVD system with ultra-high-vacuum multi-chamber (MVSystem). ... 48
Fig. 31. Schematic drawings of a) contact profilometry and b) optical profilometry using interferometry [7].50 Fig. 32. Equipment used to measure the thickness of the films. a) P-7 stylus profiler and b) ellipsometer L116 model. ... 50
Fig. 33. Scanning electron microscope (SEM) SU3500 model manufactured by Hitachi. ... 51
Fig. 34. Atomic force microscope (AFM) easyScan DFM. ... 51
Fig. 35. SEM-EDS Scios DualBeam. ... 52
Fig. 36. Spectrophotometer Lambda 3. ... 52
Fig. 37. Example of (h)1/2 against h from UV-visible measurements on an a-Si xC1-x:H film. ... 53
Fig. 38. Equipment for measurements of current-voltage versus temperature. a) Cryostat, b) Keithley 6517A electrometer and c) 331 temperature controller. ... 54
XIV
Fig. 39. AFM images of: a) the reference silicon surface, and from b) to j) a-SixC1-x:H films deposited at
110 kHz, 240 mW/cm2, 1.5 Torr, Z
H2=9. b) XCH4=0.15 and 350 °C, c) XCH4=0.35 and 350 °C, d) XCH4=0.50 and
350 °C, e) XCH4=0.70 and 350 °C, f) XCH4=0.85 and 350 °C g) XCH4=0.95 and 350 °C, h) XCH4=0.70 and
150 °C, i) XCH4=0.70 and 250 °C j) XCH4=0.70 and 300 °C. ... 57
Fig. 40. Spectrophotometer Vector 22. ... 58
Fig. 41. FTIR spectra of the a-SixC1-x:H films deposited by PECVD at 110 kHz, 240 mW/cm2, 1.5 Torr, ZH2=9.0.
P01: XCH4=0.15 and 350 °C, P02: XCH4=0.35 and 350 °C, P03: XCH4=0.50 and 350 °C, P04: XCH4=0.70 and
350 °C, P05: XCH4=0.85 and 350 °C, P06: XCH4=0.95 and 350 °C, P07: XCH4=0.70 and 150 °C, P08: XCH4=0.70
and 250 °C, P08: XCH4=0.70 and 300 °C. ... 58
Fig. 42. Transmittance spectra in the UV-visible range of the a-SixC1-x:H films deposited by PECVD at
110 kHz, 240 mW/cm2, 1.5 Torr, Z
H2=9.0. P01: XCH4=0.15 and 350 °C, P02: XCH4=0.35 and 350 °C, P03:
XCH4=0.50 and 350 °C, P04: XCH4=0.70 and 350 °C, P05: XCH4=0.85 and 350 °C, P06: XCH4=0.95 and 350 °C,
P07: XCH4=0.70 and 150 °C, P08: XCH4=0.70 and 250 °C, P08: XCH4=0.70 and 300 °C... 59
Fig. 43. Conductivity under darkness against the reciprocal of temperature of the a-SixC1-x:H films deposited
by PECVD at 110 kHz, 240 mW/cm2, 1.5 Torr, Z
H2=9.0. P01: XCH4=0.15 and 350 °C, P02: XCH4=0.35 and
350 °C, P03: XCH4=0.50 and 350 °C, P04: XCH4=0.70 and 350 °C, P05: XCH4=0.85 and 350 °C, P06: XCH4=0.95
and 350 °C, P07: XCH4=0.70 and 150 °C, P08: XCH4=0.70 and 250 °C, P09: XCH4=0.70 and 300 °C. ... 60
Fig. 44. FTIR spectra of the a-SixC1-x:H films deposited by PECVD at 13.56 MHz, 50 mW/cm2, 1.5 Torr, and
ZCH4=0.70. ... 64
Fig. 45. FTIR spectra of the a-SixC1-x:H films deposited by PECVD at 13.56 MHz, 50 mW/cm2, 1.5 Torr, and
ZCH4=0.85. ... 64
Fig. 46. FTIR spectra of the a-SixC1-x:H films deposited by PECVD at 13.56 MHz, 1.5 Torr, 200 °C and
ZCH4=0.85. ... 65
Fig. 47. FTIR spectra of the a-SixC1-x:H films deposited by PECVD at 13.56 MHz, 1.5 Torr, 200 °C, ZH2=0.90
and 25 mW/cm2 (except the film corresponding to the process P15, which was deposited at 50 mW/cm2). ... 65
Fig. 48. UV-visible spectra of the a-SixC1-x:H films deposited by PECVD at 13.56 MHz, 50 mW/cm2, 1.5 Torr,
and ZCH4=0.70. ... 66
Fig. 49. UV-visible spectra of the a-SixC1-x:H films deposited by PECVD at 13.56 MHz, 50 mW/cm2, 1.5 Torr,
and ZCH4=0.85. ... 66
Fig. 50. UV-visible spectra of the a-SixC1-x:H films deposited by PECVD at 13.56 MHz, 1.5 Torr, 200 °C and
ZCH4=0.85. ... 67
Fig. 51. UV-visible spectra of the a-SixC1-x:H films deposited by PECVD at 13.56 MHz, 1.5 Torr, 200 °C,
ZH2=0.90 and 25 mW/cm2 (except the film corresponding to the process P15, which was deposited at
50 mW/cm2). ... 67
Fig. 52. Temperature dependence of the dark-conductivity of a-SixC1-x:H films deposited by PECVD at
XV
Fig. 53. Temperature dependence of the dark-conductivity of a-SixC1-x:H films deposited by PECVD at
13.56 MHz, 1.5 Torr, 200 °C, ZH2=0.90 and 25 mW/cm2 (except the film corresponding to the process P15,
which was deposited at 50 mW/cm2)... 69
Fig. 54. FTIR spectra of the B-doped a-SixC1-x:H films deposited by PECVD at 13.56 MHz, 25 mW/cm2, 1.5
Torr, 200 °C and ZH2=0.90. ... 72
Fig. 55. UV-visible spectra of the B-doped a-SixC1-x:H films deposited by PECVD at 13.56 MHz, 25 mW/cm2,
1.5 Torr, 200 °C and ZH2=0.90. ... 73
Fig. 56. Temperature dependence of the dark-conductivity of B-doped a-SixC1-x:H films deposited by PECVD
at 13.56 MHz, 25 mW/cm2, 1.5 Torr, 200 °C and Z
H2=0.90 and. ... 74
Fig. 57. Images of the cleaning process. a) Si-wafers with resin on the films of a-SixC1-x:H after cutting,
b) cleaning of samples in the ultrasonic vibrator and c) squeeze bottles with solvent and deionized water. ... 79
Fig. 58. Protocol for thawing cryopreserved cells. a) Cells from cryopreservation, b) cell vial, c) cells thawed via water bath at 37 °C, d) preparation of cell culture media, e) 9 mL of culture medium with cells in sterile centrifuge tube and f) cells located at the bottom of the tube with culture medium. ... 80
Fig. 59. Cell culture in 75 cm2 TC flask. a) Cells without culture medium, b) cells dissolved in fresh media,
c) cells in flask ready to be cultivated, d) parameters of culture environment (incubator), e) monitoring of the cell culture using an optical microscope and f) attached cells with population above 90% confluence. ... 81
Fig. 60. Protocol to detach the cells. a) Trypsin EDTA in sterile centrifuge tubes, b) detaching of the cells using trypsin EDTA and c) detached cells after 10 minutes in incubator. ... 81
Fig. 61. Protocol to obtain the extracts of the materials. a) Samples of the materials in sample holders, b) location
of the samples within the 24-well CT plate, c) adding 267 µL of sterile medium with serum to each well, d) wells with sterile medium, including three wells without samples, e) plate seated with parafilm and f) storage
conditions in incubator. ... 82
Fig. 62. Protocol to obtain 80% of confluence within 24 hours. a) measurement of cell concentration, b) adding 1.5x104 cells to 24 wells of the 96 wells of the plate and c) storage conditions in incubator. ... 83
Fig. 63. Protocol to cultivate cells in medium with extracts. a) removing cell media from 96-well plate, b) extracting cell media with by-products from 24-well plate, d) adding 100, 70 and 50 µL of cell media with
by-products to the wells of the 96-well plate e) completing 100 µL in each well and f) 24-well plate and 96-well plate in incubator for 24 hours or 96 hours, according to the assays. ... 83
Fig. 64. Protocol for the quantification of the cell viability. a) 1X Dulbecco’s Phosphate-Buffered Saline solution, b) Trypan Blue solution, c) incorporating 10 µL of the mixture in one of the two chambers of the slide, d) inserting the slide into the slot for measurement, e) results after running the measurement and f) detailed report of the results. ... 84
Fig. 65. Images from optical microscope of cell cultures mixed with Trypsin, 1X Dulbecco’s Phosphate-Buffered Saline solution and Trypan Blue solution. a) Living cells (brown color) and b) dead cells
(blue color). ... 85
XVI
Fig. 67. Images of the control materials (baseline (BL), cupper (Cu) and gold (Au)) after 96 hours. ... 86
Fig. 68. Images of the cell cultures on the bottom of the wells after 24 hours being in contact with cell culture media contaminated with 100%, 70% and 50% of the extracts derived from the samples. ... 87
Fig. 69. Images of the cell cultures on the bottom of the wells after 96 hours being in contact with cell culture media contaminated with 100%, 70% and 50% of the extracts derived from the samples. ... 88
Fig. 70. Viability results of the assays. a) 24 hours and b) 96 hours after to be the culture cells in contact with the medium contaminated with 100%, 70% and 50% of the extracts derived of the samples. ... 89
Fig. 71. Steps for the formation of self-assembled organosilane monolayers on surfaces of a-SixC1-x:H [2]. . 91
Fig. 72. Functionalization of an a-SixC1-x:H surface using 3-aminopropyltrimethoxysilane (APTMS) as compound to form a self-assembled monolayer (SAM). ... 93
Fig. 73. Illustration of the link between glutaraldehyde and the self-assembled monolayer (SAM) of 3-aminopropyltrimethoxysilane (APTMS) on the surface of a-SixC1-x:H. ... 93
Fig. 74. Illustration of the in situ reduction of the Schiff base (imine bond) by immersing the sample in sodium borohydride (NaBH4) solution... 94
Fig. 75. Illustration of the binding of the Protein A (PrA) via glutaraldehyde (GA) as crosslinker. ... 95
Fig. 76. Immobilization of anti-E. coli polyclonal antibodies (Ab) through Protein A (PrA). ... 96
Fig. 77. Illustration of the entrapping of enterotoxigenic Escherichia coli (E. coli) by mean of a biofunctionalization layer on the surface of a-SixC1-x:H. ... 96
Fig. 78. FTIR spectra of biofunctionalization stages. S0: a-SixC1-x:H film, S1: APTMS/ a-SixC1-x:H, S2: GA/APTMS/ a-SixC1-x:H, S3: PrA/GA/APTMS/a-SixC1-x:H, S4: Ab/PrA/GA/APTMS/a-SixC1-x:H, S5: ETEC/Ab/PrA/GA/APTMS/ a-SixC1-x:H. ... 98
Fig. 79. Structure of the proposed interdigitated microelectrode array (PIMA). Illustrations of a) the top view of the array and b) the cross-section of a pair of interdigitated microelectrodes. ... 104
Fig. 80. Electron beam evaporation system used in titanium deposition. ... 105
Fig. 81. Arrangement of layers of the four Si-wafers for fabrication of the proposed interdigitated microelectrode array (PIMA) with the PECVD deposition processes a) P18 and P26, b) P19 and P26, c) P18 and P28, and d) P19 and P28. ... 106
Fig. 82. Stages of the fabrication process for the proposed interdigitated microelectrode array (PIMA). ... 106
Fig. 83. Schematics of arrays without shielding. a) Symmetric pads and b) antisymmetric pads. ... 107
Fig. 84. Schematics of arrays with shielding. a) Symmetric pads and b) antisymmetric pads. ... 108
Fig. 85. Structures of characterization. a) cross-bridge structure and b) structure of tracks. ... 108
Fig. 86. Alignment marks. a) Marks in Level 1 mask, b) marks in Level 2 mask and c) marks in Level 2 mask on marks in Level 1 mask. ... 108
Fig. 87. Photolithography procedure using the Level 1 mask. a) The layer stack on the Si-wafer, b) photoresist application, c) UV light exposure, d) photoresist development and e) top view of the array with the patterns transferred to the photoresist. ... 110
XVII
Fig. 89. Reactive ion etching characterization of a-SixC1-x:H in CF4 plasma under 50 W of power and 50 mTorr
of pressure. ... 112
Fig. 90. Layer stack on Si-wafer after B-doped a-SixC1-x:H etching. a) Cross view of a pair of microelectrodes
and b) top view of the array. ... 112
Fig. 91. Reactive Ion etching (RIE) reactor used in the Ti etching processes. ... 113
Fig. 92. Reactive ion etching (RIE) characterization of Tiin SF6, CHF3 and O2 mixture plasma... 114
Fig. 93. Layer stack on Si-wafer after Ti etching. a) Cross view of a pair of microelectrodes and b) top view of the array. ... 115
Fig. 94. Layer stack on Si-wafer after photoresist removal. a) Cross view of a pair of microelectrodes and b) top view of the array. ... 115
Fig. 95. Photolithography procedure of the Level 2 mask. a) Photoresist application, b) 3D view of the array with the photoresist deposited, c) UV light exposure, d) photoresist development (the photoresist still cover the microelectrodes) and e) 3D view of the array showing the pads without photoresist. ... 116
Fig. 96.Layer stack on Si-wafer after etching of the B-doped a-SixC1-x:H film on pads. a) Cross view of a pair
of microelectrodes and b) top view of the array. ... 116
Fig. 97. Layer stack on Si-wafer after photoresist removal. a) Cross view of a pair of microelectrodes and b) 3D view of the array. ... 117
Fig. 98. Results of each one of the stages showing the top view of one pad and a fraction of the interdigitated microelectrodes array corresponding to PIMA with shielding. a) Photolithography (Level 1 mask), b) B-doped
a-SixC1-x:H etching (array), c) Ti etching (array), d) photoresist removal in acetone, e) photoresist removal in
CF4 plasma, f) photolithography (Level 2 mask), g) B-doped a-SixC1-x:H etching (pads), h) photoresist removal
in acetone and e) photoresist removal in CF4 plasma. ... 118
Fig. 99. Alignment structures in one of the fabricated Si-wafers. ... 118
Fig. 100.SEM images of PIMAs fabricated at different finger spacing. a), b) and c) are panoramic images of PIMAs with shielding and both spacing and width of fingers of 5, 10 and 15 µm, respectively. d), e) and f) are images of interdigitated microelectrodes corresponding to the PIMAs in a), b) and c) respectively. ... 119
Fig. 101.SEM images in transverse mode. a) The cavity made using ions, b) the layers of the PIMA, including platinum, c) border of the interdigitated microelectrode, which is located from center to the right. ... 119
Fig. 102. a) Measurement terminal configuration in normal mode, b) front panel of the impedance analyzer
IM3570, c) measurement tips, manipulators and Si-wafer on triaxial chuck, d) T-connectors, measurement tips on pads of PIMAs in e) die of Si-wafer and f) in chip. ... 121
Fig. 103. a) Setting the cable length, b) and c) running open and short circuit compensations, respectively. 121
Fig. 104. Setting of the control interface and the measurement mode. ... 122
Fig. 105. Data preview window ... 122
XVIII
Fig. 107. Impedance spectra of PIMAs fabricated with the intrinsic a-SixC1-x:H layer deposited at ZH2=2.7 and
the B-doped a-SixC1-x:H layer deposited at XCH4=0.85, and with finger spacing of a) 5 µm, b) 10 µm and c)
15 µm. ... 123
Fig. 108. Impedance spectra of PIMAs fabricated with the intrinsic a-SixC1-x:H layer deposited at ZH2=9.0 and the B-doped a-SixC1-x:H layer deposited at XCH4=0.85, and with finger spacing of a) 5 µm, b) 10 µm and c) 15 µm. ... 123
Fig. 109. Impedance spectra of PIMAs fabricated with the intrinsic a-SixC1-x:H layer deposited at ZH2=2.7 and the B-doped a-SixC1-x:H layer deposited at XCH4=0.50, and with finger spacing of a) 5 µm, b) 10 µm and c) 15 µm. ... 124
Fig. 110. Impedance spectra of PIMAs fabricated with the intrinsic a-SixC1-x:H layer deposited at ZH2=9.0 and the B-doped a-SixC1-x:H layer deposited at XCH4=0.50, and with finger spacing of a) 5 µm, b) 10 µm and c) 15 µm. ... 124
Fig. 111.FTIR spectra of the intrinsic and B-doped a-SixC1-x:H films deposited by PECVD at 13.56 MHz, 25 mW/cm2, 1.5 Torr and 200 °C and used in the cytotoxicity assessments. ... 129
Fig. 112. Typical deconvoluted FTIR spectra for the intrinsic and B-doped a-SixC1-x:H films deposited by PECVD at 13.56 MHz, 25 mW/cm2, 1.5 Torr and 200 °C and used in the cytotoxicity assessments [5]. ... 130
Fig. 113. Electrical model of the PIMA outside liquid solution. ... 133
Fig. 114. Band diagram showing the space region charge of width W formed inside a p-type semiconductor when it makes contacts with a metal of lower work function. ... 134
Fig. 115. Electrical model of the PIMA outside solution including the capacitance of the space charge region. ... 134
Fig. 116. Electrical model of the PIMA outside solution including the parallel between the capacitance of the space charge region (CSCR) and the contact resistance (RTi-SiC). ... 135
Fig. 117. Impedance spectra of as-prepared and hydroxylated PIMA. ... 137
Fig. 118. Impedance magnitude spectra of biofunctionalization stages on PIMA. ... 137
XIX
LIST OF TABLES
Table 1. Lengths and bond energies of bonds present in intrinsic and doped hydrogenated amorphous silicon-carbon alloy. Data reported in the references: a [20], b [21], c [22], d [23]. ... 5
Table 2. Assignment of bonds and vibrational modes of the absorption peaks in the infrared spectra of undoped
a-SixC1-x:H films. ... 6
Table 3. Classification of some types of transducers. ... 7
Table 4. Morphological and electrical parameters of E. coli. [12] ... 30
Table 5. Capacitance per unit area of the materials involved in the development of the PIMA, water as solution and the phenomenon of the double dielectric layer. ... 31
Table 6. Design parameters of the proposed interdigitated microelectrode array (PIMA) ... 32
Table 7. Electrical parameters of materials, E. coli and medium used in the simulations. ... 34
Table 8. Deposition processes of a-SixC1-x:H thin films by PECVD at a frequency of 110 kHz, power of 800
W, chamber pressure of 1.5 Torr and ZH2 of 9.0. XCH4=[CH4]/([CH4]+[SiH4]) and ZH2=[H2]/([CH4]+[SiH4]).47
Table 9. Deposition processes of a-SixC1-x:H thin films by PECVD at RF-frequency of 13.56 kHz and chamber
pressure of 1.5 Torr. ... 49
Table 10. Data from profilometry and AFM measurements on silicon substrate (c-Si) and a-SixC1-x:H films
deposited at 110 kHz, 240 mW/cm2, 1.5 Torr, and Z
H2=9. t is the thickness, Dr is the deposition rate, Rq is the
RMS roughness, Ra is the average surface roughness and Ry is the peak-valley height. ... 55
Table 11. Chemical composition of the films deposited by PECVD at 110 kHz, 240 mW/cm2, 1.5 Torr, 350 °C,
ZH2=9.0 and at different methane-silane gas flow rate (XCH4). ... 58
Table 12. Optical band gap (Egopt), dispersión parameter (B) and E04 of the a-SixC1-x:H films deposited by PECVD at 110 kHz, 240 mW/cm2, 1.5 Torr and Z
H2=9.0. ... 60
Table 13. Activation energies (Eσ) and prefactor (σ0) of a-SixC1-x:H films for low and high temperature regimes
from electrical dark conductivity measurements in the range of 300–440 K. Process parameters: 110 kHz, 240 mW/cm2, 1.5 Torr, and Z
H2=9. ... 61
Table 14. Data from profilometry and AFM measurements on silicon substrate (c-Si) and a-SixC1-x:H films
deposited at 13.56 MHz and 1.5 Torr. t is the thickness, Dr is the deposition rate and Rq is the RMS roughness. ... 62
Table 15. Chemical composition of the a-SixC1-x:H films deposited by PECVD at 13.56 MHz, 25 mW/cm2,
1.5 Torr, 200 °C, ZH2=9.0 and at different methane-silane gas flow rate (XCH4). ... 63
Table 16. Density of Si–C bonds (NSiC), density of C–H bonds (NCH) and density of Si–H bonds (NSiH) of
undoped a-SixC1-x:H films deposited by PECVD at 13.56 MHz. ... 63
Table 17. Optical band gap (Egopt), dispersion parameter (B) and E04 of the a-SixC1-x:H films deposited by PECVD at 13.56 MHz and 1.5 Torr. ... 68
XX
Table 18. The conductivity at room temperature (σrt), the activation energies (Eσ) and the prefactors (σ0) of
a-SixC1-x:H films for low and high temperature regimes from electrical dark conductivity measurements in the
range of 300–440 K. Process parameters: 13.56 MHz, 1.5 Torr. ... 70
Table 19. Deposition processes of B-doped a-SixC1-x:H thin films deposited by PECVD at RF-frequency of
13.56 MHz, RF-power of 15 W, chamber pressure of 1.5 Torr, temperature of 200 °C and ZH2 of 9.0. ... 71
Table 20. Thickness (t), deposition rate (Dr) and root mean square roughness (Rq) of silicon substrate (c-Si) and B-doped a-SixC1-x:H thin films deposited by PECVD at RF-frequency of 13.56 MHz, RF-power of 15 W,
chamber pressure of 1.5 Torr, temperature of 200 °C and ZH2 of 9.0. ... 71
Table 21. Atomic percentage of silicon (Si), carbon (C) and boron (B) of the B-doped a-SixC1-x:H films
deposited by PECVD at 13.56 MHz, 25 mW/cm2, 1.5 Torr, 200 °C, Z
H2=9.0 and at different doping level (YB).
... 72
Table 22. Optical band gap (Egopt), dispersion parameter (B) and E04 of the B-doped a-SixC1-x:H films deposited
by PECVD at 13.56 MHz, 25 mW/cm2, 1.5 Torr, 200 °C, Z
H2=9.0 and at different doping level (YB). ... 73
Table 23. The conductivity at room temperature (σrt), the activation energies (Eσ) and the prefactors (σ0) of
a-SixC1-x:H films for low and high temperature regimes from electrical dark conductivity measurements in the
range of 300–440 K. Process parameters: 13.56 MHz, 15 W, 1.5 Torr, 200 °C and ZH2=9.0. ... 74
Table 24. Deposition parameters of the a-SixC1-x:H films used in the extract method for cytotoxicity assessment.
... 78
Table 25. Root mean square roughness (Rq) of the a-SixC1-x:H films before and after 24 hours being in contact
with cell culture media. ... 85
Table 26. Assignment of bonds and Vibrational modes of the absorption peaks in the infrared spectra of stages of the biofunctionalization process. ... 98
Table 27. Growth parameters of the thermal silicon dioxide (SiO2) layer in water vapor environment. ... 104
Table 28. Deposition parameters of intrinsic and boron-doped a-SixC1-x:H films used in the fabrication of the
four structures of proposed interdigitated microelectrode array (PIMA). The deposition time, the deposition rate and the thickness are those set and obtained for and from the fabrication process. ... 105
Table 29. Process parameter of the RIE characterization of Ti. XCHF3=[CHF3]/[SF6] and ZO2=[CHF3]/[SF6].
... 113
Table 30. Chemical composition of the intrinsic and B-doped a-SixC1-x:H films deposited by PECVD at
13.56 MHz, 25 mW/cm2, 1.5 Torr and 200 °C and used in the cytotoxicity assessments. ... 129
Table 31. Thickness (t), optical band gap (Egopt), Tauc parameter (B) and densities of Si–H, Si–C and C–H bonds (NSiH, NSiC and NCH, respectively) of the intrinsic and B-doped a-SixC1-x:H films deposited by PECVD at
13.56 MHz, 25 mW/cm2, 1.5 Torr and 200 °C and used in the cytotoxicity assessments. ... 129
Table 32. Capacitances of the PIMAs measured at 1 kHz of frequency for the three spacing finger of 5, 10 and 15 µm. ... 133
1
CHAPTER 1
Hydrogenated amorphous silicon-carbon alloy (
a
-Si
xC
1-x:H) and its
prospects in biosensors
1.
Introduction
The development of materials for biosensors involves a sequence of steps, which comprise
from the selection of the method of obtaining of the material to the tests on device. These
steps must be always oriented according to the required properties of the material. Depending
on the application, morphological, structural, chemical, optical, mechanical, electrical,
among other properties should be adjusted. The excellent mechanical, chemical, electrical
and optical properties of hydrogenated amorphous silicon-carbon alloy (a-SixC1-x:H) makes this material an excellent candidate for the fabrication of devices whose structure requires
mechanical stability, high electrical resistivity, transparency to sunlight, thermal stability and
resistance to corrosion and wear. As a matter of fact, this chapter focuses the attention on the
structure of a-SixC1-x:H and the technique to deposit it, along with the previous works in which the material has been incorporated, specifically in devices based on affinity interaction
as the immunosensors. This will allow an approach to the terminology used throughout the
manuscript.
2.
Deposition of
a
-Si
xC
1-x:H by
PECVD
Amorphous silicon-carbon alloy (a-SixC1-x) has been obtained by sputtering, laser ablation, molecular beam epitaxy (MBE), metalorganic chemical vapor deposition (MOCVD), hot wire chemical vapor deposition (HTCVD) and plasma-enhanced chemical vapor deposition (PECVD) [1]. This work uses the latter technique to deposit hydrogenated amorphous silicon-carbon alloy (a-SixC1-x:H) films, which will be studied. PECVD takes advantage of an electrical source to affect with an electromagnetic film the precursor gases in the chamber
and thus a plasma with reactive species is formed. The species in the plasma reach the
substrate surface on which the film of interest will be deposited. Among its advantages is
2
substrate surface without requiring high temperature in the substrate. The technique of
PECVD usually employs deposition temperatures between 150 °C and 400 °C, which makes it compatible with silicon manufacturing processes [2].
The deposition of a-SixC1-x:H by PECVD is carry out in a reactor with the necessary gases in the chamber of deposition. The reactor can be of two types, capacitively or inductively
coupled as is shown in Fig. 1. On the one hand, a reactor coupled capacitively consists of a
pair of flat parallel plates, with one plate operating as support for the substrate on which the
film will be deposited. On the other hand, a reactor coupled inductively is surrounded by
wire, which is part of the winding inductor, which gives rise to the electromagnetic field
inside the deposition chamber when it is excited by the supply. This work uses two different
reactors coupled capacitively and more details will be given in chapter 3. About the gases,
the process requires silane (SiH4) and methane (CH4) or acetylene (C2H2) as precursor gases,
diborane (B2H6) or phosphine (PH3) as dopant gas and argon (Ar) and/or hydrogen (H2) as
dilution gas.
a) b)
Fig. 1.PECVD reactor coupled a) inductively and b) capacitively [3].
There are seven parameters that vary during a deposition by PECVD: the frequency and power density of the source, the chamber pressure, the substrate temperature, the flow rate
of precursor gases, the dilution rate and the doping level. The electrical source usually
operates at radio frequency of 13.56 MHz, although other frequencies may also be used and
3
species depends on the power density provided by the source. In this regard, the power of the
source regulates the ratio of deposit and defines the mechanism by which takes place the
deposition [5]. The pressure in the chamber during deposition influences the density of
reactive species and hence the deposition rate; under low pressure, although the reactive
species are more energetic owing to its longer mean free path, the density of species in the
plasma is lower, which reduces the deposition rate. Moreover, it has been reported that at low
pressures (below 800 mTorr) the films have low uniformity [6]. The substrate temperature,
instead, affects directly the morphology of the film. Increasing the substrate temperature, the
kinetic energy for migration of absorbed molecules increases, and thus the reactive species
can find the site energetically more favorable during deposition, improving the structure of
the film (lower degree of disorder) and reducing the residual stress. At this point it is
noteworthy that as the substrate temperature increases, the hydrogen promotes the conversion
of free radicals in molecules [7], and simultaneously varies its incidence due to desorption
from the surface [8]; therefore, not necessarily an increase in temperature will increase the
deposition rate and the conductivity of the film. The composition of the film is controlled by
the ratio of precursor gases (defined as the ratio between the flow of methane or acetylene,
and the total flow of precursor gases). For example, if the methane-silane gas flow rate (XCH4)
is increased, the percentage of carbon incorporation into the films will be higher [5, 9–10].
Furthermore, the dilution rate of hydrogen (ZH2) (defined as the ratio between hydrogen flow
and the flow of precursor gases) takes part in the dynamics of bond formation during the
deposition of the films, influencing not only on the deposition parameters but also on the
properties of the films [11–13]. Finally, the electrical conductivity of the films can be
enhanced by the doping level (Y) (defined as the ratio between the flow of dopant gas and
the total flow of precursor gases, given in percentage) [9, 14, 16].
3.
a
-Si
xC
1-x:H structure
The understanding of the structure of hydrogenated amorphous silicon-carbon alloy is closely
linked to the meaning of each one of the words that identify it. Starting with the adjective
"amorphous", which excludes it from crystalline materials. a-SixC1-x:H is a solid material that is in a state outside thermodynamic equilibrium and has no spatial order in the long range
4
[16–17]. About the composition, amorphous silicon carbon consists of carbon and silicon
atoms, but when it is deposited by PECVD becomes hydrogenated, due to hydrogen incorporated during deposition from precursor and dilution gases.
The atomic arrangement of a-SixC1-x:H is better understood from the structure of carbon and silicon carbide (SiC). As is shown in Fig. 2, carbon forms mainly two kinds of local
structures: the diamond structure composed of three-dimensionally spread sp3 bonds and graphite structure composed of two-dimensionally spread sp2 bonds. Instead, as is shown in Fig. 3, SiC forms a structure like diamond composed of sp3 bonds and although theoretical analysis has evidenced that it could form a structure like graphite, it has not been obtained
experimental yet [18–19]. In the case of a-SixC1-x:H, depending on the ratio between the densities of sp3 bonds and sp2 bonds, the structure may be diamond-like or graphite-like. An illustration of the structure of a-SixC1-x:H is shown in Fig. 4. This atomic arrangement is not only due to its structure, which is neither diamond-like nor graphite-like, but also to its
amorphous nature, which allows slight variations in lengths and angles of the bonds (see
Table 1).
a) b)
Fig. 2. Atomic structures of carbon as a) graphite and b) diamond [20].
a) b)
5
Fig. 4. Atomic structures of hydrogenated amorphous silicon-carbon alloy [22].
Table 1. Lengths and bond energies of bonds present in intrinsic and doped hydrogenated amorphous silicon-carbon alloy. Data reported in the references: a [23], b [24], c [25], d [26].
Bond Bond length (pm) Bond Energy (kJ/mol)
H–H 74.2a 432.00±0.04a
Si–Si (c-Si) 235.2a 222a
C–C 154a 345.6a
Si–C (SiC) 185a 318a
Si–H 148a 318a
C–H 109a 411±7a
B–H 119a 345.2±2.5b
Si–B 200c 317±12b
C–B (B4C) 144b 372a
P–H 144a 341d
Si–P (SiPH5) 225d 363.6b
C–P 184a 264a
Molecule Bond length (pm) Bond Energy (kJ/mol)
CH4 109a 439.3±0.4b
SiH4 148a 377.8±5.0b
B2H6
BH3–BH3 133 150.6±12.6b
B2H5–H 119 ≤429.7b
PH3 144a 351.0±2.1b
One of the measurement techniques most widely used to study the bond structure of a
material is FTIR spectroscopy. In this technique, an incident light beam in the infrared range strikes the sample surface, while a detector measures the spectrum of reflected or transmitted
6
series of peaks can be observed at different wavenumbers, which constitute the fingerprint
of the material. The Fig. 5 shows a typical spectrum of a-SixC1-x:H ranging from 500 cm-1 to 3500 cm-1, whereas the Table 2 depicts the assignment of the peaks and the vibrational mode of the bonds.
500 1000 1500 2000 2500 3000 3500 0 2 4 6 8 10 12 14 16 18 20 Si-H 2 Si-H 2 Si-CH n C-CH 3 CHn Si-C Ab sorban ce ( a. u. )
Wavenumber (cm-1)
Si-H
n
C=C
CHn
Fig. 5.FTIR spectrum of hydrogenated amorphous silicon-carbon alloy.
Table 2. Assignment of bonds and vibrational modes of the absorption peaks in the infrared spectra of undoped
a-SixC1-x:H films.
Bond Vibrational mode Wavenumber (cm-1) References
Si–Hn Wagging 630–650 [7, 27]
Si–C Stretching 650–700 [28] Si–C, Si–CHn Stretching, wagging 740–800 [7, 27–30]
Si–H2 Rocking, scissors 845, 895 [27]
CHn, Si–CH3 Wagging, bending 1000, 1245 [7, 27, 28]
C–CH3, CHn Bending, scissors 1370, 1450 [27]
C=C Stretching 1540, 1630 [27, 30] Si–H2 Stretching 2080 [7, 27, 28, 30]
7
4.
Biosensors
A biosensor is a device that integrates the biochemical recognition of biological compounds
(proteins, DNA, viruses, bacteria, cells, etc.) with a signal transducer [31]. Therefore, the
specification of a biosensor comprises the recognition method and the transduction method.
When any of these elements is changed, the biosensor changes, then there are as many
biosensors as there are possible combinations of recognition and transduction methods.
Among the most common recognition methods are ion recognition, affinity interaction,
nucleic acid hybridization, enzymatic action and recognition using cells and tissues of
biological origin. The list of transducers, instead, is a bit longer and starts by differentiating
between free-label and label-based transducers. In biosensors in which any compound in the
recognition process is not detectable, other compound can be used to label one or most
product compounds and to do them detectable by the corresponding transducer. In this case
the transducer is known as label-based. In any other case, the transducer is label-free. Some
of the main transducer used in biosensors are classified in Table 3.
Table 3. Classification of some types of transducers.
Transduction method Transducer
Calorimetric (free-label) Thermistor, microthermopile and microcalorimeter, among others.
Mechanical
(free-label, mass detection)
Piezoresistives
Quartz crystal microbalance (QCM) Based on surface acoustic waves (SAW)
MEM devices Microcantilever
Electrical (free-label) Resistive, capacitive and impedance-based
Based on semiconductor electronic devices, ion-sensitive
Magnetics (free-label) Based on magnetic nanoparticles
Electrochemical (free-label) Potenciometric, amperometric, conductometric and impedimetric
Optical
Free-label Label-based
Based on surface plasmon resonance Interferometric and reflectometric
Raman and FTIR spectroscopy Based on photonic devices
Based on fiber optics
Fluorescent Chemiluminescent
Luminescent Radioactive
8
Since Leland C. Clark, the father of biosensors, developed in the early 60’s a device that used
the enzyme glucose oxidase (GOx), coupled to an electrode, as sensing element for
measuring the concentration of glucose in the blood or in other fluids or gases [32], there has
been a growing interest in biosensors. However, today the ratio between devices that have
become commercial and those others developed is low. And although both application areas
and transduction methods have been on the rise, the requirements so that a biosensor fulfills
the expectations in an application have not been an easy task. Areas such as diagnosis and
prevention of diseases, food industry, control of water quality and environmental pollution,
agriculture and since the beginning of the XXI century the defense against biological attacks
require the development of biosensors with high sensitivity, high precision and short
response time, label-free, configurable for various diagnoses, of easy operation, low cost and
low degradation in biological medium [33–34].
5.
Applications of
a
-Si
xC
1-x:H in biosensors
In the last 20 years, looking for new materials that improve the performance of biosensors and allow implantation into living organisms for long periods of time, a-SixC1-x:H has emerged as one of the candidates [34]. In terms of compatibility with current manufacturing technology based on silicon, a-SixC1-x:H is deposited at low temperatures, which do not affect the previous stages of the manufacturing process [2]. As for the function within the structure of a biosensor, the properties of a-SixC1-x:H can be adjusted with the deposition parameters, giving versatility to the design [5, 9–10]. In addition, a-SixC1-x:H is chemically stable and its surface can be biofunctionalized [6, 35].
One of the functions that has so far fulfilled a-SixC1-x:H biosensor is as light filter [36–38]. Lipovšek et al. [36] performed depositions of a-SixC1-x:H films varying the gas flow ratio
(C2H4/[C2H4+SiH4]) from 0% to 99% to obtain films with the greater rejection ratio between
excitation wavelengths and emission wavelengths of some important fluorescent molecules
(specifically some amino acids, coenzymes and vitamins). These filters are inserted between
the fluorophores (fluorescent molecules) and a photodiode and are used to reduce the noise
caused by the excitation light in the intensity of light detected by the photodiode. Conde et
9
Jóskowiak et al. [38] were a little further and designed an array using three different filters
based on a-SixC1-x:H to simultaneously detect tryptophan (Trp), dinucleotide nicotinamide adenine in its reduced form (NADH) and dinucleotide flavin adenine in its oxidized form
(FAD).
In order to take advantage of the increased stability of organic monolayers linked through
covalent Si–C bonds, some studies have incorporated thin films of a-SixC1-x:H as an interface deposited on metal [35, 39–40]. Touahir et al. [39], for example, optimized the layer
thickness of a-SixC1-x:H deposited on 200 nm aluminum (acting as reflective layer) that should be used in fluorescent microarrays to achieve maximum sensitivity. With a similar
purpose, Galopin et al. [35] made use of the advantages of the covalent bond between the
surface of a-SixC1-x:H and organic monolayers to improve the efficiency of sensing through localized surface plasmon resonance. For this purpose, they deposited a a-SixC1-x:H thin film on gold nanoparticles, achieving not only covalent bonding, but also a larger sensing area,
since the area between nanoparticles also is functionalized. According to the study by
Touahir et al. [40], a layer of ~5 nm of a-SixC1-x:H between metallic nanostructures and a monolayer ended in a carboxylic group (–COOH) allows a maximum sensitivity in the
detection of hybridization of DNA using localized surface plasmon-enhanced fluorescence
spectroscopy.
Other efforts have been taken towards the implementation of a-SixC1-x:H as a coating for implantable biosensors [29, 41–42]. Boltz et al. [41], in their work published in 1996, set out
the requirements of a material acting as a coating for stent. According to those requirements,
doped a-SixC1-x:H with high dilution in hydrogen, through its semiconductive nature, the wide band gap of mobility, the possibility of achieving conductivities close to 104 Ω/cm and of obtaining low density states within this range, can be a candidate to reduce degradation
due to thrombogenesis. Although Boltz et al. not presented in their publication a
characterization of the material, its findings on studies of cytotoxicity, hemolysis, and blood
compatibility tests in patients were satisfactory. Also, Cogan et al. [42] have evaluated the
dissolution rate of the material in saline phosphate buffer (PBS, pH 7.4), obtaining a
dissolution rate of 0.1 nm/h to 90 °C, being it lower than that of other materials of the
