Nano-structured silicon based lab on a chip for diagnostics Karpiuk A.D., Starodub N.F.
Luchenko A.I., Melnichenko M.M.
Laboratory of biosensors National University of Life and Environmental Sciences of Ukraine Kiev, Ukraine
[email protected];
[email protected]
Physics Department Taras Shevchenko National University of Kiev Kiev, Ukraine
[email protected]
Abstract—The immune biosensor are proposed in the lab on a chip form based on the nano-structured silicon for simultaneous analysis of samples number. The procedure of the nanostructured silicon formation and preparation of the photoresistor-test strips have been described and used in case of the cow retroviral leukemia express diagnostics. Keywords—nano-structured silicon; lab on a chip; immune biosensor; diagnostics; retroviral leukemia.
I. INTRODUCTION Nano-porous (nano-structured) silicon has a number of functional characteristics: high adsorption capacity due to the highly developed surface, the ability to change the energy state of the surface by the variable of the size of the nanocrystals, air voids, porosity, the nature and composition of the interphase boundaries. Currently, sensors are designed to determine the concentration of various gases, alcohol, moisture, some biological molecules and toxic elements, based on suppression of photoluminescence of nano-porous silicon, interferometer sensors and the effects of thermo acoustic abilities [1-7]. However, other physical and chemical effects of nanoporous silicon are poorly explored, the knowledge of which will promote to create a lab on a chip in order to increase their accuracy, stability, speed, reliability determining the concentration of various substances and ensure the necessary level of safety of human health and the environment. Now existing designs "lab-on-a-chip" is a high-tech and highprecision medical instruments. Nanotechnology is allowed to create very small specialized system. "Lab-on-a-chip" can contain hundreds or even thousands of analyzers to test various chemical compounds and their derivatives. Thus, the times when it is necessary to perform testing for a long series of numerous analyzes are fading. In a case, such laboratory is able to analyze different samples at the same time and the time of analysis is shortened up to 15-30 minutes. Last simplifies and reduces the cost of biochemical diagnostics. For today silicon (monocrystalline, polycrystalline, amorphous, porous) is the main material of microelectronics and it presents a great interest for a new developing direction on the basis of using of quantum size effects. The application
of different modifications of silicon to create nano-electronics and nano-photonics devices put forward a number of innovative requirements for most processes and methods of control parameters as processes and new generation of the created instruments and devices. The basis of the direction of creating of nano-structured films of silicon is the results of work at the creation and using of its porous forms. At the formation of the nano-structured silicon changes not only the structural properties, which leads to a change in band gap and the emergence of quantum size effects, but also the formation of new compounds on the surface of silicon with high hydrogen content and amorphous silicon. Such a complex structure leads to new electro, photovoltaic, thermal, electroluminescent and photo luminescent properties allowing to create new types of semiconductor devices biosensors. Formally, nano-structured (nano-porous) materials can be regarded as nano-composite, in which holes plays the role of the second phase, randomly or regularly distributed in the matrix. Nano-structured silicon is known to be a biocompatible material with advanced and chemically development surface. However, free and available surface for interaction with gases and solutions may be achieved up to 1000 m2/g. Among the methods of obtaining of nano-porous silicon layers the most common are: anodic electrolytic etching of single crystal silicon in the solutions based on the hydrofluoric acid application and chemical etching. The first allows a wide range of properties change of nano-porous silicon layers, but has some significant drawbacks, among them are: the complexity of communication with standard silicon technology, partial using of the area of the plate, the inability of mass processing. Thus the properties of nano-porous silicon obtained by the chemical etching of mono-crystalline silicon, in terms of the application as a multi-functional material are virtually unknown. Chemical etching method makes it possible to grow thin films of nano-structured silicon and requires no special equipment; it is simple and high technological, which allows easy adaptation to the conditions of industrial production.
II. EXPERIMENTAL RESULTS The nano-structured silicon plate were formed from monocrystalline substrates by the surface modification with chemical etching in a solution of hydrofluoric and nitric acid. We used single-crystal silicon wafer grown by the Czochralski method proposed for the manufacture of solar cells. Original plates were characterized by district p-type conductivity (boron doped), resistivity of 1 Ohm/cm, (100) crystallographic orientation, thickness of 350 mm. The thickness of nanostructured silicon ranged from 3 to 60 nm. This parameter was controlled during process of the cultivation of chemically modified silicon layer. Image of surface analysis (Fig. 1) obtained by scanning tunneling microscopy suggests that the resulting nano-structured silicon surface has a fractal nature. Lateral dimensions of the crystallites are several nanometers with height about 20 nm. In addition, there is queasy regular location pores, and the size of individual pores are large fluctuations, and their diameter varies in depth pores.
compounds on the surface of nanostructures. The latter one can be polysilanes, siloxan with different oxygen, hydrogen and hydroxyl groups, as well as oxides. These chemical groups may correspond components of SiH, SiO, SiOH, SiF, Si2O. Study of hydrogen content in the surface and distribution of its concentration in the depth of nano-structured silicon films have showed that atomic hydrogen penetrates in the silicon wafer, where it effectively passivity's defects at grain boundaries and in the bulk, reducing the recombination of minority carriers. Thus, the results of secondary ion mass and Auger electron spectroscopy's show that all samples of nano-structured silicon formed are nano-composite with nano-scale silicon crystals surrounded by oxidative phase SiOX, hydrogen, oxygen and carbon. Obtaining of samples of high homogeneity nanostructured silicon in the composition of the sample area and good reproducibility in terms of chemical contents. On the basis of nano-structured silicon samples were made photo resistors (Fig. 2a) in the form of test strips. Contacts with thickness of 3 mm were formed from aluminum through a mask by magnetron sputtering. In experiments it was chosen direct physical adsorption of biological material because it has soft connection method that preserves the biological activity of biomolecules.
Al nanostructured silicon singl crystal silicon 10 mm
Fig. 1. Three-dimensional view of chemical modified surface of monocrystalline silicon (field of scanning 0,5 * 0,5 µm ).
For obtaining the information about the chemical composition of the surface of the nano-structured films, as well as about their thickness, the samples was effected by the silicon layered etching as well as argon ion beam spectra measurements by Auger electron spectroscopy and secondary ion mass spectroscopy. Analysis of Auger spectra of sample showed that C, O, Si and SiOX were as the main elements, which enriched the surface. The content of O, C and SiOX is 49.6 at.%, 24.1 at.% and 25.8 at.%, respectively. The results obtained by the secondary ion mass spectroscopy were in good agreement with Auger data analysis. The most intense peaks in the mass spectra of secondary ion surface nano-structured silicon in the comparison with the no-treated one is H+, C2+, SiH+, C2H6+, K+, SiO+, SiOH+, SiF+, Si2O+ for positive and H-, C-, CH-, O-, F-, C2-, C2H2- for negative ions. The obtained results of secondary ion mass spectroscopy showed that the range of observed ions may be associated with the remnants of the chemical etchant on the surface and in the pores (H+, H-, ОH-, O-, F-), as well as components of
5 mm
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I, mA
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Ab
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Fig. 2. Photoresistor-test strip (а) and sensor responses in form of changes of photocurrent at the immobilization of antigen (Ag) and antibodies (Ab) (b).
Investigated biological object were deposited in volume 1 µl on the photoresistor surface area between contacts (Fig. 2a) and than was dried in air conditions at the room temperature. In the proposed biosensor it was analyzed changes of the photosensitivity nano-structured silicon measuring photocurrent at a voltage of 5 V. Measurements showed that the dark current of the sensor is virtually unchanged and the photocurrent after application of antigen and antibody
increases considerably (Fig. 2b). Further on the basis of nanostructured silicon samples a mini laboratory on a chip (Fig. 3a) which consists of two independent produced biosensors. To test the proposed lab on a chip we studied the interaction of immune complexes (Ag-Ab at the diagnostics of retroviral leukemia) on the nano-structured silicon surface. The surface of one photoresistor with the already immobilized antigen was contacted with the solution of specific Ab inpositive serum blood. Simultaneous the surface of another photoresistor was interacted with negative serum blood which had not specific Ab. After that the surfaces of photoresistors were completely dried in air conditions. The changes of photocurrent are shown in Fig. 3b.
diagnostics for one animal but for several ones too. It was demonstrated that the described approach based on the nanostructured silicon realizes practice demands in the respect of sensitivity, selectivity and simplicity of analysis fulfilment. The efficiency of the proposed approach was compared with the traditional standard ELISA-method. It was revealed that this biosensor give possibility to have the same sensitivity but the overall time of analyse was shortened in more than 10 times with less expenses. Now we have already the preliminary results about the possibility of the diagnostics of the diabetic states of patients in the respect of simultaneous control of the total immunoglobulin's, anti-insulin antibodies, glucose level and others parameters. This approach demonstrated high efficiency at the express control of such diacethylated polyamines as spermine and spermidine at the express biochemical diagnostics of breast cancer.
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I, mA Ab+
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Fig. 3. Test-strip of lab on a chip (а) and sensor responses at the interaction of specific complex of Ag-Ab at the diagnostics of retroviral leucosis (b). Left - specific Ab was presented in solution to be analysed and right - absent.
The obtained results allow to state that during the application of positive serum the immune complex is formed and there is a sharp increase in the photocurrent. While applying negative serum it was not found virtually nothing. It was mentioned that the photocurrent was changed for higher during 1-3 hours (Fig. 4). It was reflected the kinetic of the immune complex formation. 1200
I, mA
Ab+
800 Ag 0
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Fig. 4. The kinetic of the immune complex formation.
This procedure of analysis was fully realized by us for an express diagnostics of retroviral leucosis when level of the viral specific antibodies should be discovered in blood or in milk of ill cows. There is possibility not only fulfilling
III. CONCLUSIONS Thus, nano-structured silicon is very promising material for the development not only optical devices such as lightemitting diodes, photovoltaic cells and optical filters, but biosensors meet modern requirements, such as ease of use, inexpensive, high precision, selectivity and speed of analysis, and miniaturization requirements. The sensor is a versatile device that works in a laboratory and in the field. Test strips have characteristics according to the necessary requirements analysis and is variable element. The developed biosensor based on nano-structured silicon is very promising for use in so-called systems of lab on a chip because it can be applied for rapid analysis of various immune responses and is fully compatible with silicon planar technology used in the manufacture of the semiconductor devices. The developed type of biosensor was effectively used for retroviral leukemia diagnostics as well as its applicability was preliminary confirmed at the control of level of development of the diabetic state and breast cancer. REFERENCES [1] C. Baratto, G. Faglia, E. Comini, G. Sberveglieri, A. Taroni, La Ferrara V., L. Quercia, G. Francia, “A novel porous silicon sensor for detection of sub-ppm NO2 concentrations,” Sensors and Actuators B: Chemical, – № 77, 2001, pp. 62–66. [2] A. Шмырева, Н. Мельниченко, “Сенсорные системы с применением нанопористого кремния,” Электроника и связь, Ч.1, 2006, c.17–22. [3] N. Starodub, V. Starodub, “Biosensors based on the photoluminescence of porous silicon,” Application for environment monitoring, Sensor electronics and microsystem technologies, № 1, 2005, pp. 63–74. [4] Ivo Rendina, Ilaria Rea, Lucia Rotiroti, Luca De Stefano, “Porous siliconbased optical biosensors and biochips,” Physica E, 38, 2007, pp. 188–192. [5] Min-Jung Song, Dong-Hwa Yun, Joon-Hyung Jin, Nam-Ki Min and SukIn Hong, “Comparison of Effective Working Electrode Areas on Planar and Porous Silicon Substrates for Cholesterol Biosensor,” Japanese Journal of Applied Physics, Vol.45, № 9A, 2006, pp. 7197–7202. [6] N. Lorraina, M. Hiraouia, M. Guendouza, L. Hajia, “Functionalization control of porous silicon optical structures using reflectance spectra modeling for biosensing applications,” Materials Science and Engineering, B 176, 2011, pp. 1047– 1053. [7] A. Rossi, L. Wang, V. Reipa, Th.E. Murphya, “Porous silicon biosensor for detection of viruses,” Biosensors and Bioelectronics, 23, 2007, pp. 741–745
