Abstract
Nanostructured materials possess better tunability of their properties compared to their bulk counterparts. These properties have opened up new avenues for fabricating highly sensitive, miniaturised and cost effective sensing devices. For the realisation of a rapid and easy to use immunosensor, nanostructured porous silicon/polyaniline heterostructure has been prepared electrochemically which provides label free, real time electrical detection with high sensitivity for biomolecules (mouse IgG/goat antimouse IgG) from whole serum. Nanostructured thin film SnO2 sensors prepared by Langmuir Blodgett technique has been investigated for room temperature gas sensing under various chemically polluting ambiances (NH3, H2S, SO2, CO, H2, NO2 and CH4) using electrochemical methods which were able to detect specific gases at room temperature in the presence of other residual gases.
Introduction
Biochemical sensors and sensing systems are currently active areas of research as they pertain to design and fabrication of devices that allow simple and rapid real time detection of biological or chemical molecules with high selectivity and sensitivity in complex samples. A direct immunosensor that does not rely on the use of a detectable label could lead to simpler assay formats and ideally lower times of detection. Electrochemical devices allow generation of clinically acceptable data with inexpensive pocket sized instruments suitable for over-the-counter sales. These sensors allow one to design portable units, conduct field trials, on-line measurements with multi-analytic analysis, direct, cheaper and faster measurements. Research on electrochemical immunosensors started with those built on Si/SiO2. Electrochemical immunosensors are affinity sensors that use antibody as the recognition element and the diagnostic immunochemical (antigen-antibody) reaction coupled to an electrochemical transducer. Affinity based sensors are devices in which the chemical or biological agent is selectively bound to the surface through a specialised surface coating. The capacity changes at the electrode–electrolyte interface as a result of the formation of antigen antibody complexes on the electrode were applied to develop a series of capacitive affinity sensors. Amperometric detection of antigen antibody interactions was accomplished by the application of redox modified antigens or antibodies and their competitive association to the electrode support in the presence of analyte–antigen or antibody. The sensitivity, selectivity, quantification and time response of these affinity based sensors are functions of the substrate material, specialised surface coatings and signal transduction methods used.1–4 Porous silicon (PS) has all the advantages of silicon: it is easy to miniaturise and has low cost.5–8 The enhanced surface area due to the porous structure, would result in an increase in the measured signal strength which would allow scaling down of the sensor area. Thus it is an ideal candidate for the fabrication of nanoscale biochemical sensors leading to rapid and real time monitoring with high sensitivity and reusability.9–15
To prevent or minimise the damage caused by atmospheric pollution, monitoring and controlling systems are needed that can rapidly and reliably detect and quantify pollution sources within the range of the regulating standard values. Metal oxides used as sensing materials in semiconductor gas sensors (ZnO, TiO2, or SnO2) have a wide band gap, typical for insulators. They possess conductivity in the range of semiconductors due to point defects (oxygen vacancy) in the crystal structure which causes physisorption or chemisorptions of surface species such as water vapour and oxygen. SnO2 thin films are known to detect reducing gases with high sensitivity and reversibility at high temperature. Detection of target gases is generally carried out by measurement of dc resistance. Traces of foreign gases (H2S, NH3, CO, NO2) affect the adsorbed oxygen coverage on SnO2 film, which lead to a change in film resistance. Surface adsorbed species (O−, O2−, OH among which O2− is the predominant species at room temperature) on SnO2 thin/thick film surface interact with reducing gases at high temperature leading to desorption of oxygen resulting in an increase in the conductivity of the film.16–18 With oxidising gases conduction band electrons are further removed resulting in a decrease in the conductivity of the film. Though these sensors are highly sensitive, the selectivity is not very good at low temperature. Gas sensors for detecting air pollutants must be able to operate stably under deleterious conditions, including chemical and/or thermal attack with high selectivity and sensitivity. Therefore there is constant search for reliable, wearable or hand held gas sensors which can operate at room temperature. The selective and sensitive detection of polluting gases (NH3, SO2 and NO2) at room temperature have been successfully achieved using nanostructured SnO2 thin films prepared by Langmuir Blodgett (LB) technique.19–23 Here a brief review of biochemical sensors based on nanostructured materials such as: PS based biosensor and hand held toxic gas sensor operating at room temperature are presented. Prototyping of hand held toxic gas sensor with alarm has been demonstrated for trace levels of NH3 gas.
Experimental
Fabrication of PS immunosensor
In the present study, electrochemical etching of p-type single crystalline silicon with ρ 1–10 Ω cm was done using current density of 32 mA cm−2. The electrolyte used is the combination of hydrogen fluoride (HF) and absolute ethanol in the ratio 1:1. PS of different porosity, structural morphology etc. was obtained by changing the parameters of electrochemical etching.13–15 This low thermal budget process would allow integrated systems with capacitive immunosensors to be processed by downstream processes post integrated circuit fabrication. Anodically oxidised PS was treated with aminopropyltriethoxysilane (APTES) which helps in covalent immobilisation of biomolecules through a bifunctional coupler, gluteraldehyde. The detailed description of immunosensor fabrication is given elsewhere.
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Figure 1 shows the standardised protocols required for the fabrication and characterisation of PS based capacitive immunosensor.
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The immunosensor was constituted using the functionalised silicon/PS as the working electrode, platinum plate and wire as counter and pseudo reference electrodes, phosphate buffered saline as electrolyte. Potentiostat/Galvanostat PGSTAT20, Echochemie, the Netherlands, was used for electrochemical experiments and impedance measurements.
Various process steps (anodic oxidation, aminopropyltriethoxysilane (APTES) interaction and gluteraldehyde coupling) required for covalent immobilisation of antibody on PS interface
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Fabrication of nanocrystalline SnO2 thin film sensor
Nanocrystalline SnO2 thin films on quartz substrates were prepared using Langmuir–Blodgett technique. The detailed procedure is described elsewhere. 20 After heating the as-prepared LB films (2 × 1.5 cm2) at 600°C in air, nanostructured SnO2 thin films were obtained. Sensors were prepared by depositing interdigitated gold electrodes using a shadow mask (five electrodes each side) with 0.5 mm electrode width and 0.2 mm separation between the electrodes on nanocrystalline SnO2 thin film. A small gas testing chamber for continuous on-site monitoring was made of brass (5 cm diameter, capacity 30 ml) cylindrical chamber with perforations where the gas can be injected so that the natural diffusion of gas takes place. Connectors were taken from the contact pads on the sensor film and the sensor is placed in the gas testing chamber. Known amount of various gases (NH3, NO2 and H2O) were injected using gas tight syringes. Sensor response of the films towards the target gases have been obtained by current vs. time measurement using Keithley electrometer. All measurements were carried out at room temperature (25°C) and at atmospheric pressure without any carrier gas flow.
Results and discussion
PS based immunosensor
PS is a nanocrystalline material, which is obtained either by electrochemical or stain etching of monocrystalline silicon. The porous structure mainly consists of silicon nanowires/channels and dots. We have prepared two types of columnar porous structures: thin cylindrical columnar macroporous silicon (silicon column width few microns, length ∼50 μm and few tens of microns pore width) and short irregular columns with cylindrical pores (pore/column width ∼ few tens of microns) as the substrate for immunosensor.13–15
Considering the immunosensor as an electrochemical capacitor, capacitance transients and capacitance voltage measurements have been used for the detection of biomolecules. Characterisation of immunosensors based on polished silicon and PS have been carried out after each process step and are described elsewhere.13,14 Binding of specific target molecules to the immobilised probe molecules inside the pores can cause a measurable change in capacitance due to the change in the dielectric constant and thickness. A large change in capacitance is observed after specific interaction of analyte–antigen (goat antimouse IgG) with antibody (mouse IgG) which is covalently immobilised on PS. About one order increase in response was observed for PS based immunosensors compared to polished silicon immunosensors. These devices had fast response and were found to be stable and reusable. 13
In another approach PS/polyaniline (PS/PANI) heterostructure has been prepared electrochemically which provides label free, real time electrical detection with high sensitivity for the specific model biomolecules (mouse IgG/goat antimouse IgG) (Fig. 2). PANI is conducting polymer which is also an organic semiconductor, processible in various oxidation states or doping conditions having dramatic changes in electrical conductivity depending on the state. It has got naturally occurring NH groups, which can bind covalently with biomolecules. The sensor structures based on PS/PANI were prepared easily by directly immobilising the biomolecule through gluteraldehyde coupler. These biosensor structures made by inexpensive methods were used to detect specific biomolecule down to at least a picomolar concentration range and were found to be reusable.
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Figure 3 represents the typical capacitance response measured at 1 kHz for the mouse IgG immobilised PS/PANI sensor in the presence of whole serum containing goat antimouse IgG. Using static capacitance measurements the sensor showed a change in capacitance of 0.55 µF for 300 µg of goat antimouse IgG in 1160 µl whole serum. Repeated measurements indicate that these sensors can be used for detection of specific biomolecules directly from whole serum with high sensitivity. High sensitivity and easy processability of PS/PANI structures could be understood by the change in surface charge of the silicon channels in the PS structure that occurs by PANI deposition while providing covalent binding with biomolecules with its naturally occurring NH groups. The space charge layer in the PS/PANI channels get further modified by the surface charge change created by the specific analyte–antibody binding, giving rise to high sensitivity. It was found that a substrate bias in the accumulation region of PS gives the optimum sensitivity and specificity.
Typical capacitance response measured at 1 kHz for the mouse IgG immobilised PS/PANI sensor in the presence of whole serum containing goat antimouse IgG. Triangular symbols represent the capacitance voltage response with 250 µg of antibody mouse IgG and square symbols represent the capacitance voltage response with 300 µg of goat antimouse IgG present in the whole serum
Nanostructured SnO2 thin film sensor for room temperature toxic gas sensing
The physical properties limiting sensor devices fabricated in polycrystalline thin film can be readily overcome by exploiting nanoscale structures. In the case of polycrystalline thin films sample resistance measured using dc current has contributions from different regions of the sample such as intragrain, grain boundaries and electrode sample interface. The effect of grain boundary barrier can be reduced by using nanostructures. The effect of grain size and grain boundary barrier on carrier density modulation (Δne) is schematically shown in Fig. 4 (ne vs. grain thickness l), for micro and nanogranular films. The two cases are scaled using Debye length LD of SnO2 for a given temperature for micro and nanogranular films. For polycrystalline thin films with the thicknesses l> 2LD (LD is the Debye length for SnO2 at a given temperature, l is the film thickness), oxygen chemisorptions induces a band bending only near the surface. In contrast, the effect of surface adsorption on nanostructures (LD on nanograins >l/2) is to change the location of the Fermi level within the band gap of the nanostructure causing an appreciable conductance drop while decreasing the effect of grain boundary barrier.17,21 In order to exploit the nanostructure properties we have deposited ultrathin films with LD>l/2 by controlling the thickness of SnO2 films using LB technique.
Schematic diagrams of space charge formation at the grain boundaries (upper part) and carrier density modulation (lower part) due to surface adsorbed species (O−, O2−) on micro and nano granular film. LD is Debye length, l is the grain size, ne is carrier density, Δne change in carrier density © Elsevier21,23
We have fabricated SnO2 nanocrystalline thin films by Langmuir Blodgett (LB) method, which have shown reliable and highly sensitive detection of trace gases such as NH3, SO2, NO2 and H2S gases at room temperature with high specificity.19–23 Recovery within 30 minutes was observed for these sensors without any carrier gas flow after NH3, SO2, and H2S injections. XPS studies19,20 on the thin SnO2 films have indicated the presence of oxygen vacancies on the surface with about 31 weight for adsorbed oxygen related surface states indicating a large influence on the surface properties. Estimation from XRD data and FESEM micrographs has shown the size of the nanocrystallites as 10–20 nm.19,23 The carrier concentration calculated from Mott–Schottky plot is ∼4 × 1016 cm−3
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which gives Debye length of ∼12 nm. Since the grain size is less than 2LD there is no effect of grain boundary barrier. Absence of grain boundary barriers and charge carrier depleted nanograins of the SnO2 thin films support the room temperature response and high sensitivity towards the trace gases. These sensors were tested for humidity effects which confirmed that these sensors were not affected by relative humidity upto 98 and show the same sensitivity for a period of one year under closed conditions. Figure 5 shows the sensor setup with interdigitated electrode coated SnO2 thin film, gas sensing chamber and the electronic alarm. The response of the sensor towards repeated injections of 10 ppm NH3 gas is shown in Fig. 6.
The sensor setup with interdigitated electrode coated SnO2 thin film, gas sensing chamber and the electronic alarm The response of sensor towards repeated injections of 10 ppm NH3 gas
Conclusions
Two typical nanostructured semiconductor devices, PS/PANI heterostructure and SnO2 thin films, have been prepared and established as highly sensitive immunosensors and room temperature operating gas sensors, respectively. These two devices have been compared with their macrostructure counterparts in sensitivity and ease of operation and were found to be better choices in terms of sensitivity, ease of use, reliability and operating temperature. Porous nano structures are particularly interesting with its tunable structure and size for very sensitive, selective detection, separation and quantification of various analytes. For integrated systems low temperature processing of PS enables processing after CMOS fabrication. Very small grain size and high surface-to-volume ratio associated with the nanocrystallites in the SnO2 thin films allow the chemical sensors to be operated in the most sensitive, grain-controlled mode at room temperature.
Highlights
Immunosensor using nanostructured porous silicon/polyaniline heterostructure has been prepared.
Transient capacitance detection of 260 µg mL−1 goat antimouse IgG from whole serum.
Room temperature ammonia detection (10 ppm) using nanostructured tin oxide film.
High sensitive and reliable biochemical sensing by nanostructured thin films.
Footnotes
Acknowledgements
I would like to express my sincere thanks to my collaborators, Dr Sipra Choudhury and Ms K. G. Girija, BARC, India for providing the SnO2 thin films and the electronic alarm units respectively for the realisation of gas sensor.