S.C. BAYLISS, L.D. BUCKBERRY and A. MAYNE Solid State Research Centre and Biomaterials Research Group De Montfort University Leicester LEI 9BH UK
1. The growing use of porous silicon in bioapplications
Porous silicon has now been used in a range of in vitro and in vivo bioapplications, from implant coatings to cell culture substrates and biosensors [1-5]. These widening applications are in part due to the large specific surface of porous silicon, the nano and micro-machinability of silicon, and the potential of nano and microstructured silicon to be used in novel devices due to the greatly modifiable optoelectronic properties. However there is also a growing body of literature which testifies the biocompatibility of porous silicon. Thus there appears to be many exciting biological areas in which porous silicon could play a major role. These include medical, environmental and computing technologies, and generally will require the immobilization of cells and enzymes in specific regions on porous silicon substrates. Once this has been achieved, the porous silicon can be built into, or supplied with, the relevant device architectures. However much background work needs to be done for these first steps to be realised. This itself requiring an understanding at the molecular level of interactions at the porous silicon surface. This interface between a cell and an inorganic material may seem straightforward at the micron level. It is in fact a dynamic system at the molecular level. Firstly, the substrate usually exhibits an oxide on the surface, due to exposure to air and the sterilization pre-treatment. Secondly, it is in a physiological environment, and generally proteins in the serum will coat the surface. In addition to this, it is necessary to ensure that the correct biosystem is adhered to the surface at the correct location. To go further in the deployment of porous silicon, therefore, it is necessary first to gain an understanding of the current techniques used in the research and development of biomaterials. And it is necessary to assess their applicability for the case of porous silicon. In particular the requirements for biomaterials need to be described, and the methods for controlling the interactions of biomolecules with biomaterials need to be clarified.
Biomaterials are materials which show biocompatibility. As well as for inert implants, such materials can be used in devices for restoring sensory and/or motor function, living skin equivalents, drug delivery systems, sensors, and for supporting matrices [6]. The term biocompatible includes the effect of the substrate on the function of the biosystem, including lack of toxicity, and the effect of the biosystem on the function of the biomaterial, including its sensitivity and stability. Along with the growing body of literature describing use of porous silicon for bioapplications, a recent study of toxicity of the silica and silicic acid forms of silicon following the MTT assay method of Buckberry has been reported [7]. Respiring cells metabolise MTT to its formazan derivative, a reaction catalysed by succinate hydrogenase. The derivative is purple, and the dye released from adhered cells cultured in the MTT spiked media can be assessed spectrophotonically. A sigmoid response is expected at a specific concentration, and the concentration which kills 50% of the cells (corresponding to 50% absorbance of untreated cells) can be used to calculate toxic efficacy. The B50s have, however, shown a definite lack of toxicity (Figure 1). The cell line chosen for our studies is the immortalised B50 rat hippocampal neuron cells, as these cells produce neurotransmitters necessary for the neuron-like functions, as would normal glial cells. The B50s were exposed to a range of concentrations from 0.0001 to 100 mM of either chemical for a period of 24hrs. The substrates used were coverslips since it was known that these substrates would not produce metabolites which could compromise the data. No toxicity was observed from either the silica or the silicic acid: cell cultures remained 100% viable (as compared with a control culture) over the exposure period of 24 h (Figure 1) even at the highest concentration levels used. Of course the in vivo or in vitro environments exposed to PS wafer would not include concentrations as high as 100 mM of silicic acid and silica: it is at these concentrations that salts precipitate out.
MTT assay
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Figure I. Toxicity of silicic acid and silica in culture ofB50 cells on silica, using MTT assay.
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The observation of lack of toxicity is supported by the anecdotal evidence of cell growth directly onto PS [8] and by other works [9].
3. Culturing cells in vitro
Recently there have been major advances in the techniques available for the in vitro culture of cells derived from mammalian tissues. Along with improvements in cell handling methods, the developments which have given rise to this progress include cell line characterisation and isolation, and identification of specific cellular growth factors. This has led to the situation where it is now possible to culture almost any type of mammalian cells.
Since there are 2 different types of cell systems, cells which grow in suspension and those which are anchorage-dependent, different methods are required for cell culture. For applications where interfaces between cells and inorganic materials are at issue, as in the deployment of porous silicon for implants, sensors and cell culture substrates, we are interested in the latter type. Individual cells from such cultures adhere to the substrate and also make contact with neighboring cells. Apart from ensuring that the correct physiological environment, including adhesion proteins (usually in the serum, or applied to the substrate surface), is in place, that the substrate is compatible with the biological system, and that the cell culture is healthy, no further chemical modification is required. The serum contains the correct concentration of nutrients and oxygen for the cells to grow and reproduce. Note that certain substrates (and certain cell lines) do not require adhesion proteins - in particular porous silicon has been successfully used to culture 4 cell line types without the need for such adhesive. In the case of enzymes, microbial cells and antibodies, however, the process of immobilization requires chemical modification.
4. Controlling cell growth and division
The growth of cells on surfaces has led to an increased understanding of the cell function and life cycle, and of the differentiation between healthy and cancerous cells. Furthermore the effect of drugs, toxins, and growth hormones on cell function can be investigated. In the case of porous silicon substrates, neuronal growth factor (NGF) has been tested on B50 cell cultures [10]. The reason for choice ofNGF is that we hoped to slow down the cell division rate, thereby allowing individual cells to have increased longevity. This is of importance when one is trying to produce devices based on addressing specific cells - the problem with immortalized lines such as B50s is that they thrive very well on certain surfaces.
This means that they become confluent after a few days, eventually leading to the inability of the cells on the support surface to acquire the necessary nutrients to support their metabolism. This in tum leads to apoptosis, a form of intemallyprogrammed cell death, effectively destroying the device characteristics. As an
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alternative to addition of factors such as NGF, a primary cell line could be used, but use ofNGF is much simpler.
Cell size vs NGF concentration (96 h post-seeding)
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Concentration (11M)
Figure 2: Length and width ofB50 neurons cultured with 3 concentrations ofNGF.
NGF-supplemented DMEM with a range ofNGF concentrations from O.l to 0.75mM was supplied to cells in culture on porous silicon and on coverslips over a period of 96 hours. The cells were then fixed in glutaraldehyde at 24, 48, 72 and 96 hours, goldcoated, and imaged using SEM. The effect of NGF on the cell morphology has been assessed through the average length and width of the cell body (Fig. 2), and by cell counting.
There is a monotonic decrease in average cellular length and width with increase in NGF concentration but at present it is not known whether these effects are accompanied by changes in cell vitality and function, and in particular, the ability to process signals. In other words the biochemical effect ofNGF on B50 cells is unclear.
The cell count data show that compared to a standard the NGF produced no increase in cell number, but also no decrease even with the highest concentration supplied. Once again an understanding at the molecular level is required. Studies are underway which seek to (i) confirm that cell division is suspended, and (ii) confirm if the changes in morphology are due to a decrease in cell viability. This would ultimately result in apoptopic cellular 'rounding', release from the substrate and death. Alternatively, this may be an intermediate morphological state prior to redifferentiation.
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5. Effect of porous silicon surface features on cell anchorage
In addition to the nutrient requirements for cells to be viable on a surface, surface topology is of great importance [11-14]. These effects are found to be more dramatic for feature sizes around and below the sizes of the external cell structure (some, axons etc.). Thus, although it is clear that B50 cells are viable on porous silicon without the usual requirement of an adhesive layer, we need to determine whether such cells have any preference for surface features on a nm-micron scale. As a preliminary investigation, by lithography we have produced a regularly array of plateaux and troughs in porous silicon separated by 100 micron (Figure 3), and have estimated the preference for adhesion points through cell counts: for an average of 15 images, cell counts numbers were- plateaux 65, troughs 32, ridges 98. Thus B50 cells appear to prefer to adhere to the ridges, possibly due to the presence of increased surface charges at these points.
Using fluorescence interference contrast microscopy, it has been estimated by the Fromherz group [15] that cells do not attach directly to a surface support, but rather sit up to 100nm above the surface. This has great implications for direct electrical addressing of neuron cells, and will be discussed below.
Figure 3: left: B50s cultured on an array of porous silicon plateaux and troughs; right: side view, showing good interconnections between B50s cultured on porous silicon, and the presence of anchorage supports from
the cell to the surface.
6. Immobilization techniques
There are at present a range of general techniques used for immobilization of cells and enzymes. These are 1. Adsorption and covalent coupling (used for enzymes) [16] 2. Metallinklchelation processes (used for biocatalysts) [17,18] 3. Gel entrapment (used for cells and enzymes) 4. Microencapsulation (used for enzymes)
These methods make possible a wide range of applications, such as production of immobilized enzyme electrodes [19] and electrochemical techniques [20], which could
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be used to drive specific biochemical reactions. The ability to immobilize cells could give rise to completely different and important developments, such as the transforming of steroids, manufacture of cell factories for synthesising specific products, and novel bioassays.
The technique chosen for immobilisation is based on whether anyone mode of attachment will disrupt the structure of the protein and hence its activity. It is also important to avoid overloading to prevent steric hindrance which will reduce the activity by reducing the binding.
(a) Adsorption and covalent coupling Adsorption is the simplest method. The procedure for this is i. Mix enzyme or other protein and support ii. Incubate iii. Centrifuge to separate soluble from insoluble
The disadvantage is that the enzyme is not bonded to the support but adsorbed (via saltlinkages), so changes in pH, ionic strength and temperature could debind the enzyme.
Examples of current adsorption solid supports are alumina, calcium carbonate collagen, cellulose, glass (porous), hydroxyapatite, silica gel. It is possible that porous silicon could be used as the solid support in this method.
(b) Covalent binding It is important that the amino acids involved in the activity of the protein are not bound to the solid support (binding of this type is minimised if the enzyme involved is bound in the presence of its substrate). To ensure this, covalent modification is used. Typical supports for covalent modification are agarose (sepharose), dextran (sephadex, a sugar polymer), glass and polyacrylamide co-polymers.
For this the support must first be activated, and the most widely used activated agent is cyanogen bromide (which is bound to the support) as no activation step is required. The cyanogen bromide then reacts with free amino groups of proteins.
(c) Metallinklchelation processes U sing chelation processes [17,18] should be simpler and quicker than using covalent modification as this latter method requires matrix activation as an extra step. Good candidate metals for this have been titanium and zirconium (particularly good because the oxides are non-toxic). Increased activation of a range of enzymes on various supports have been found including the transition metal chloride compared to that in its absence.
The method generally used is: i. titanium (III) chloride is used as a reducing agent for the nitroaryl derivative of cellulose. ii. the resulting aminoaryl derivative promotes the diazo coupling of the protein.
The result should be direct binding between the hydroxide and hydrogen terminations on the porous silicon and those in biomolecules in the cell membrane. Thus this will reduce the physical gap between the cell/enzyme and the porous silicon to a few well-defined mono layers.
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7. Biocomputing
The possibility of the production of a well-defined interface between silicon and neurons could be used as the basis for novel computing systems based on conventional binary data processing interfaced to living parallel computational units. For this the dynamic nature of the interface must be taken into account.
By chance the sizes of the comparable units involved in these 2 types of data processing, the soma of the neuron and the transistor chip, are rather similar, being on the order of microns. Even the relative signals are similar and of the same order of I V. The main difference between the systems is that neural systems are capable of training and adaptation. In addition, neural systems exhibit low power consumption, (40W for a sleeping human including cell regeneration). Although each action potential travels relatively slowly, there are upto thousands of connections to each neuron in the brain, and over hundreds of thousands of neurons. Thus there exists in the brain a highly complex 3D architecture, which would be impossible to reproduce in an artificial network in the foreseeable future. This enables a living system to be extremely efficient at pattern recognition, even in the presence of highly damaged input data.
The analogue computer, on the other hand, is capable of highly precise calculations, but if parts of the processor are damaged, the whole system breaks down. There are great gains to be had, therefore, by a combined approach to computational tasks.
Towards this end there have been many attempts to address individual neurons using conventional semiconductor structures. Very exciting results have been obtained by the Fromherz group in Munich [21] on silicon, and the Jerry Pines group in Caltech [22] on gold/silica. Both of these groups have demonstrated electrical stimulation and recording. The Pine group have further packaged individual neurons in units arranged in 4x4 arrays with nutrient supply available. However, even with a comprehensive enclosure and food supply, the neurons behave plastically, attempting to grow out of their containers. Furthermore the signals recorded are much weaker than would be expected (of the order of ~V rather than mY). Once these problems have been solved, then much study on neural signal processing can take place, and a wide range of computational devices can be realised, such as the pRAM developed by the Clarkson group in London[23].
As an alternative to the approaches above, we have carried out mechanical stimulation of B50 cells using a microprobe [24]. The cells were cultured on a 22mm coverslip, and were loaded with Ca+ dye. It was possible to record the passage of a signal through the cell network 15 mm away from the stimulation point. The signal was recorded as a function of time, and the signal pulse peak was obtained 30 microseconds after stimulation. No signal was recorded when an artificial break was made in the network, showing that the signal was propagating through the network.
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8. Final remarks
Earlier work on cell stimulation used the patch-clamp method to stimulate and probe a cell response [25]. This is not suitable for long-term studies, as the neuron quickly dies, and furthermore it is not investigating the neuron under natural conditions. The procedures described above [21,22] are an improvement as they use external electrodes. Despite the additional progress in the cell culture procedures and the design of neuron addressing devices such as the Neurochip, it is surprising that good signal to noise data on stimulation and recording has not been achieved. It may be that the neuron's apparent resistance to toxicity is linked to its inability to transmit artificial signals. This is perhaps a self-protection mechanism which allows only the correctly-produced information to be passed. Thus either mechanisms other than electrical may need to be developed, or the functionality itself may need to be adapted at a biomolecular level.
Finally it should be noted that cells can adapt to their environment over time, sometime involving a change in functionality. Although it is possible to revive some of these functions, using the appropriate physiological environments, it is possible that in a living system, such as is envisaged for biocomputing, the neurons will be modified over time. In this case it may not be possible to predict how the system will behave, and this leads to many questions concerning the use of living systems for technological reasons.
Thus, despite the problems highlighted on signal processing and cell localization, it appears porous silicon has a lot to offer to the area of neurobiology as it so far given no indication of toxicity to neurons, and good adherence when cultured on porous silicon. In addition it can be patterned using conventional methods and has optoelectonic emission and detection properties. The development of a silicon to neuron interface is of particular importance for the development of energy efficient, powerful 3D architectures, complementing or even replacing the need for artificial intelligence. We apologize for not including in this paper more of the vast body of work in the area of neural stimulation.
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