Biosensors – how to detect bacteria and counterfeits

We often perceive the electronic and the biological as two different approaches. However, for Professor Ilona Piekarz's work to make sense, both of these worlds need to combine, otherwise the biosensor is not going to work.

Currently, laboratory methods are the ones applied the most for bacteria detection. A smear taken from the patient goes into the hands of specialists who, firstly, make sure the bacteria have the right conditions to proliferate and, after some time, they observe under the microscope whether bacteria is in fact there. To interpret an image from under the microscope, one needs a qualified staff and that is the factor that significantly limits the efficiency of this method.

When using biosensors, the samples could theoretically be examined by unqualified people. A sample could be applied onto the biosensor whose signal would go via a scanner to a computer. The right software could process the signal automatically, so that the user could read a ready-made piece of information on the result. From the user's perspective, glucometers work in a similar way, a drop of blood is placed on a special test strip which is inserted into the device. Only a few seconds later, we may see the amount of glucose in blood displayed on the screen.

The closer to bacteria, the better

Professor Ilona Piekarz from the Institute of Electronics at the Faculty of Computer Science, Electronics, and Communications works among others on biosensors which detect Escherichia coli, bacteria commonly found in the lower intestine. The research has been initiated by Professor Sławomir Gruszczyński from the said institute who was the supervisor of Professor Piekarz's doctoral thesis. One of such devices developed at the AGH University takes advantage of microwaves, i.e. radio waves of micro length. Using the frequency range with the electrical properties of bacteria having the greatest variability increases the chance of their detection.

One of the devices intended for detecting bacteria was made from Low Temperature Co-fired Ceramic (LTCC). It is a technique that allows to fabricate small structures from layers of ceramics of various thickness bonded under high pressure and co-fired. As a result, in between them, we may fit elements essential for the construction of a sensor which should not be isolated from external factors, like the electric circuit power. Our "access" to the system is the highly sensitive element of the sensor made of metals biocompatible with bacteria. One of them is gold on which a test sample is placed. The probes connected to it conduct a signal, which then goes to the vector analyser, the measuring instrument. Subsequently, the scientists proceed with calculations to be able to conclude whether the signal has changed (so whether it has detected something it was supposed to) or whether it remained the same (which may indicate a negative result).

For the signal to be interpreted properly, each biosensor needs to be optimised to detect specific bacteria. Covering the sensor with a special bio-sensitive layer provides certainty that any changes in the signal are caused by the presence of E.coli rather than of other species or impurities. In this case, the layer consists of Escherichia coli antigens, as a result of which only this particular type of bacteria my stick to the layer. Responsible for the development of this layer, a group of scientists from the Institute of Immunology and Experimental Therapy of the Polish Academy of Sciences has supported the AGH University scholars in the implementation of similar interdisciplinary projects. After covering the sensor with a selective layer, the researchers verify whether the sensor does not react without any bacteria or for any other types of bacteria.

What is more, for the signal to change, the electrode, so the metal with bacteria on its surface, must have a particular pattern. For example, unless it is wrapped properly, the bacteria will not be able to bind to the sensitive area and, as a result, they will be found only on an area which will not provide a change in signal.

The result mentioned above was obtained owing to a biosensor, the design of which was described in the prestigious Biosensors and Bioelectronics journal. It was significantly smaller than the LTCC biosensor and the dimensions of sensitive elements slightly exceeded the size of E.coli. The circuit was made with the use of monolithic microwave integrated circuit technology, one that enables obtaining details of micro and nano size but also has a matrix of sensors instead of a single sensitive element. Although the researchers did not manage to go below the detection threshold mentioned, they see a great potential for even better results with this type of sensors. Research is already underway on the development of a biosensor with a very high ratio of sensitive area to total surface area, which significantly increases the probability of detecting bacteria and thus potentially lowers the detection threshold.

On the trail of sweet counterfeits

What could we use such electric sensors for? For instance, for unveiling scams in the food industry. One of the most counterfeited food products is honey, as consumers are frequently unaware of the fact that a product with such a label may not be fully natural, but contain artificial syrups. As this difference is not detectable in taste, the counterfeit rarely comes to light. However, it may pose difficulties to some, for example to the diabetics. Meanwhile, if you want to know the ingredients of a product, you need to send a sample to a lab and wait for the result. AGH University Associate Professor Ilona Piekarz is currently working on a sensor which would make it possible to determine whether a product is natural honey or a honey-like product containing some artificial additives. For the consumers to be able to use such devices for their own purposes, such sensors are developed with the use of cheap materials, like laminate. At the AGH University, the implementation of such projects is possible only with the application of rapid prototyping, like 3D printing and laser micromachining of printed circuits. Since recently, the AGH University Institute of Electronics has been equipped with LPKF ProtoLaser U4, a laser system for creating conductive traces with minimal details, in the range of tens of micrometres, not only on laminates but also on other inorganic materials, what is particularly useful for further (bio)sensor research. The incremental and hybrid methods for creating microwave circuits are a domain of AGH University Associate Professor Jakub Sorocki from the Institute of Electronics, who has collaborated with Professor Piekarz for many years now.

There will be time for speed

To enhance the biosensors which are to detect bacteria even further, it is essential to create more and more versions of them. Meanwhile, the development of a physical iteration is more technologically demanding than for circuits that can be made on a laminate. For the biosensor to be sensitive to bacteria, its surface must be perfectly smooth. A regular laminate, although cheap, is not suitable for tests with such small elements like bacteria, which would fall into the protrusions on its rough surface and would become impossible to detect. To carry out a project with LTCC technology or integrated technology, the AGH University researchers cooperate with foreign centres, meaning high costs and long waiting time, therefore, testing a designed circuit or introduced changes is not possible at a short notice. The prize is dictated by the production of a production model of the sensor. Mass production of sensors could be economical, but producing a few of them only for research purposes is quite costly. That is the reason why biosensors on glass are being developed at the AGH University Academic Centre for Materials and Nanotechnology, to improve the biosensor prototyping and testing procedure and limit the production costs.

Effective biosensors, ones that would enable real-time bacteria detection, would for sure compete with optical methods. Although they work on higher frequencies and are thus more sensitive, they require the application of markers, frequently of radioactive nature. Thus, they do not remove the problem of demanding preparations, the need to use laboratories and involve qualified personnel.

The current research has been focused on maximising the sensitivity of biosensors, both as regards the microwave construction as well as the bio-sensitive layer. Another step is to design an automatic data processing system for the implementation of sensors to common use. In the near future, the work will commence also in this direction.

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