In the NanoNextNL program a number of projects was started and lead to innovative science and application. Below you will find a number of examples of projects from the NanoNextNL program.
Drop of blood reveals heart infarct
A woman arrives at the accident and emergency department with chest pain. A simple finger prick could be enough to diagnose a heart infarct within a few minutes. That can be done with the Minicare I-20, which Philips will launch on the Benelux market in mid-2016. ‘This device is the result of ten years of intensive research,’ says research scientist Matthias Irmscher.
A tube of blood is now standardly taken from patients who arrive at the hospital with chest pain. The blood goes to a laboratory. About one hour later the result is known and this indicates whether the patient has a heart infarct. Meanwhile the patient is kept under observation and waits in anxiety and uncertainty. In 15 to 30% of cases that is not necessary. For example, the patient has muscle pain, joint inflammation or angina pectoris, an annoying condition but not life threatening.
Based on a single drop of blood, the Minicare system provides the result within a few minutes. ‘We measure the presence of the protein troponin,’ explains Irmscher. That protein is released if heart cells die due to a lack of oxygen.
Minicare is tested at Catharina Ziekenhuis in Eindhoven, NL (video)
Faster, simpler, more cost-effective
The Minicare I-20 fits in the trend towards point-of-care medical applications. ‘We develop systems that are faster, simpler and more cost-effective than the current laboratory tests. We not only want to speed up clinical decisions but also achieve higher efficiency and quality within healthcare. That applies not just to hospitals but to other locations as well. For example, general practitioners will be able to test for more things themselves and establish a diagnosis straightaway.’
No prior knowledge needed
New medical technology must be simple to operate, cheap to use and provide a quick and accurate result, says Irmscher. ‘The result must be comparable with that of the current time-intensive laboratory tests. You must be able to operate the technology without any prior knowledge, for example, without needing to know how to take a blood sample from a vein. We work with a finger prick of blood, which is a volume of just 20 to 30 microliters. This means we need to be able to measure very low concentrations of the protein. That was our greatest challenge.’
The product can be operated by people with little specific prior knowledge: you stick a plastic cartridge in the readout device and place a drop of blood on it. Within a few minutes the device tells you whether the protein troponin is present in the blood and if it is, in what quantity.
Within Philips we are working together with universities and other companies on a further expansion of the possibilities. We want to be able to measure several different proteins so that psychological disorders or brain damage, for example, can also be diagnosed with a finger prick of blood.
When Minicare I-20 is launched on the market, the work on the technology will certainly not be finished yet. ‘Within Philips we are now working together with universities and other companies on a further expansion of the possibilities,’ states research scientist Irmscher. ‘We want to be able to measure several different proteins so that psychological disorders or brain damage, for example, can also be diagnosed with a finger prick of blood. And we want to further improve the sensitivity as well.’
The cartridge of the Minicare I-20 consists of a piece of plastic that contains very small liquid channels that lead the blood to a chamber. The chamber contains the secret: tiny magnetic spheres that are covered with antibodies. As soon as a troponin protein flows past such a sphere the antibodies capture it.
Minicare is based on the use of magnetic nano particles (video)
Steering with magnets
The readout device contains magnets with which the magnetic spheres can be moved. As soon as the spheres have been able to bind enough troponin molecules, they are drawn to the bottom of the cartridge with the help the magnets. The base is also covered with antibodies and so the troponin molecules bind to the base as well. Subsequently the number of spheres attached to the base is taken as a measure for the concentration of troponin molecules in the blood drop. If the non-bound spheres are subsequently drawn upwards by a magnet then only the bound spheres remain on the base. To measure the number of spheres, the base is illuminated from below with light. The amount of shadow on the base finally indicates the quantity of troponin present in the blood.
Measuring minimal concentrations
‘With the magnetic spheres we therefore ensure that the proteins are bound as quickly as possible. With the so-called ‘rinsing step’, we ensure that non-bound spheres are pulled off the base thereby reducing the noise in the measurements. Thanks to these two steps, the sensitivity of this detection method is far better than that of other rapid troponin tests,’ explains Irmscher. ‘With this approach we can measure substances at concentrations of 30 billionths of a gram per litre.’
As the test works with full blood, a number of hurdles had to be overcome. ‘Ultimately we measure the protein concentration in the blood plasma and so we had to develop a filter that separates the plasma from the red and white blood cells.’ That was not easy, explains Irmscher. ‘You need to produce a filter that holds blood cells, for example, while allowing through the proteins you are interested in. The filter must not become clogged up and it must work with very different blood compositions.’
It was therefore pointless working with model liquids, he says. ‘Right from the start we worked with full blood from many different individual patients. The variation in the quantity of red blood cells between different patients is enormous. Producing a filter that is flexible enough to cope with all those extremes was therefore a considerable challenge. Within NanoNextNL researchers produced a theoretical model for that filter.’
Fundamental research has played an important role in the development of the final product, he says. ‘For example within NanoNextNL, a test setup was developed so that we could observe exactly what happened with the spheres. It was like a cross-section of the chamber with an observation window in it so that we could follow the movements of the spheres. This allowed us, for example, to determine the optimum concentration of the spheres. You must have enough spheres in the solution to bind as much of the protein as possible. However, if you use too many spheres then they obstruct each other’s movement and they cannot bind to the base and be detected.’
Label on cancer cell for targeted treatment
Producing biological molecules to kill cancer cells without damaging the surrounding healthy tissue. That is the ultimate aim of the company Tagworks Pharmaceuticals, a start-up that has emerged from the NanoNextNL programme.
Existing treatments against cancer often have serious side effects. Chemotherapy, for example, is targeted at cells that quickly grow and divide. Healthy cells, however, can also grow and divide quickly and so hair loss and damage to the stomach wall are some of the side effects. Also in the case of radiation treatment it is still very difficult to target just the tumour tissue. To limit the amount of healthy tissue damaged, nuclear medicine physicians often use a lower dose than is desirable for the complete destruction of the tumour.
Pointer to the tumour
The start-up Tagworks Pharmaceuticals uses chemically modified antibodies that recognise tumour cells. After an injection they attach themselves to the surface of the cancer cell and therefore act as a pointer to it. By subsequently injecting a different molecule that selectively binds to the modified antibody you can specifically aim radioactive emitters or drugs at the tumour tissue. You can also use this technique to very specifically image the tumour and any possible metastases. In that case you attach a source that emits gamma radiation to the second molecule. This radiation can be imaged using a scanner.
As antibodies that do not bind the tumour will remain in the body for a long time, it is not always possible to immediately bring the radioactive emitters to tumours in the first step, says founder and CEO Marc Robillard. Then the radioactive substances would remain in the body for far too long. Sometime after the first injection, a second injection is needed with the radioactive substances. ‘Our radioactive molecule binds via a rapid click reaction to the label on the tumour only. Anything that does not bind is quickly urinated out of the body. With this approach we can achieve doses ten times higher than is the case for current drugs without any severe side effects or an accumulation of radiation.’
Tagworks recently expanded its application possibilities with a new discovery. They have succeeded in not only attaching the molecules to the label but also removing these from the label in a controlled manner. This discovery now makes it possible to very specifically attach medicines to a tumour using the antibodies and to remove them again at will. With this approach, for example, you could apply chemotherapy to just the cancer cells and that would drastically reduce the side effects. Studies are now being performed on mice with intestinal cancer.
Robillard is so convinced about his product that at the end of 2011 he started his business with a considerable part of his own money. In addition he received a grant from NanoNextNL and he has a licence on several patents. The seed for the company was planted in 2004 at Philips, which produces medical scanners. To make diseases such as cancer more visible, Philips started a lab to produce biomolecules and nanoparticles. Those molecules led in turn to research into possible treatment methods. When Philips decided to stop this line of research in 2010, Robillard wanted to go further and started his own company.
Robillard’s company is participating in NanoNextNL programme 3C, molecular imaging: ‘Thanks to NanoNextNL I have access to an entire consortium. And I don’t have to pay these people. NanoNextNL is therefore a fantastic asset for starters like me.’
Video shows DNA in action
How does a piece of DNA become a person? What happens at the DNA level if somebody becomes ill? To be able to answer such questions, start-up LUMICKS developed the first microscope in the world that can show in real-time how DNA and proteins respond to each other at the molecular level. NanoNextNL programme director Gijs Wuite from the programme 8B was one of the founding fathers of this microscope.
Cancer, diabetes, muscular diseases or cystic fibrosis: errors in the DNA form the underlying cause of a range of diseases. To be able to understand what goes wrong and when it would be fantastic if you could observe the processes in which DNA and proteins interact with each other at the molecular level. That is exactly what can be done now thanks to a combination of techniques that start-up LUMICKS has incorporated into a microscope named C-TRAP.
A second product even makes it possible to manipulate and measure thousands of molecules at once. Thanks to that this Acoustic Force Microscopy technique is suitable for point-of-care diagnostics, such as measuring glucose levels with a finger prick, says Olivier Heyning, CEO of LUMICKS. ‘Now in some cases it takes two weeks for a specialised lab to analyse blood samples. Our technology can be integrated into a handy device that can recognise certain viruses within a few seconds, for example, without the need for any cells to be cultured.’
From static to dynamic
‘Up until now scientists have mainly worked with tools that can study static situations’, says Heyning about the development of the C-TRAP. ‘From a biological point of view it is very valuable to be able to follow exactly what happens during the interaction between molecules.’ The research group of Gijs Wuite and Erwin Peterman from VU University has worked for more than ten years on techniques such as optical tweezers – with which focused beams of laser rays are used to hold an object – fluorescence microscopy and systems to manipulate fluid at the microscale. Via LUMICKS, a combination of these techniques has now become available as the product C-TRAP, which can be used by other researchers and the pharmaceutical industry.
Thousands at once
‘With our combination of optical tweezers, fluorescence microscopy and microfluidics we saw for the first time exactly what happens with DNA when it is organised by proteins, a process which you previously needed extensive modelling work for’, says Heyning. Fantastic of course but still not good enough. Scientists must be able to make thousands of measurements of identical molecules for their results to be statistically significant. ‘Therefore the group of Wuite and Peterman has developed a new way of being able to manipulate and measure thousands of molecules at once.’
By pushing away the red beads with ultrasound, the force needed to stretch the molecules can be measured.
Manipulating with sound
The so-called Acoustic Force Microscopy (AFS) method works using ultrasound. Each molecule to be investigated is affixed on one side to a glass plate and on the other side to a small plastic ball. With the help of ultrasound researchers can push away the balls in a controlled manner and note the force required to stretch or break the molecule. This gives an insight into the structure and functioning of the molecules.
‘We are now busy testing different prototypes together with clients. Up until now they are all very enthusiastic’, says Heyning. The company also has a healthy financial outlook for the time being. The start-up recently received a Take-off grant from Technology Foundation STW, a Horizon 2020 Future Emerging Technologies (FET) Open Grant from the EU and financial support from the NanoNextNL Valorisation Programme to bring the AFS technique to the market. In addition, the first systems have already been installed at a university in Göttingen and at the Harvard Medical School.
Cell membrane on a chip
Researchers Séverine Le Gac and Verena Stimberg have developed an artificial cell membrane on a chip. This chip can be used, for example, to study how medicines, cosmetics or nanoparticles can influence human cells. And that can be done without the need for laboratory animals.
From an initial inventory that Stimberg made in the framework of the Risk Analysis and Technology Assessment chapter of her PhD thesis, it transpired that the artificial cell membrane had various potential applications. With the chip it is possible to test the effect of cosmetics or medicines without the need for laboratory animals. The chip is also suitable for checking if certain nanoparticles are harmful to human cells.
The chip simulates, in particular, the ion channels in a cell membrane. Ion channels are proteins in the cell membrane, which ensure that salt ions can flow in and out of the cell. That is important for bodily functions such as movement, thinking and feeling. If something goes wrong with an ion channel then that can lead to severe diseases such as epilepsy, cardiac arrhythmias or cystic fibrosis. More than ten percent of the medicines on the market are therefore specifically aimed at these ion channels.
Disadvantages of the current method
To test these medicines it is necessary to determine how they influence the ion channel concerned. The current ways of testing this either measure the effect on the ion channel indirectly or are time-consuming and expensive. They also require living cells and therefore a specialised laboratory needs to perform the tests.
Researchers Verena Stimberg (left) and Séverine Le Gac (photo: Gijs Ouwerkerk)
In a NanoNextNL project, researchers from the University of Twente have developed a chip of approximately 1 cm x 2 cm in size, which consists of two glass plates with a Teflon layer in between. In the Teflon layer a hole of about 100 micrometres (one tenth of a millimetre) in diameter is made. The glass plates contain micro-channels that are connected with each other via the hole in the Teflon. Lipid and then aqueous solutions are rinsed through the micro-channels. As a result of this a membrane is spontaneously formed in the hole in the Teflon that serves as a model for the cell membrane.
Cheaper and quicker
‘We have equipped the chip with both optical and electrical measurement techniques’, says Stimberg. ‘This makes it possible to measure the effect of the proteins or molecules added on the properties of the cell membrane.’ By adding a substance to be tested, such as medicines or chemicals, it can be directly and indirectly checked if this influences the transport of ions through the protein channel. As the chip works with small quantities of the substances to be tested, the tests could be cheaper than the current methods. Two companies were interested in this technology and together with Le Gac and her team they have further developed several aspects of the chip in a new project.
Detecting pneumonia quickly and cheaply
A small device that can detect bacteria in exhaled air. With that you could quickly establish if somebody is suffering from a respiratory infection. Michel Klerks from the company Innosieve and Marien de Jonge from RadboudUMC have received a Valorisation Grant for this from NanoNextNL.
Developing a new device that can cheaply establish a diagnosis for respiratory infections within a couple of hours. That is the ambition of Michel Klerks and Marien de Jonge. Respiratory infections can develop into pneumonia within two days. Pneumonia can be life-threatening, especially for patients who are already suffering from respiratory disorders such as COPD or cystic fibrosis.
The early and rapid detection of the bacteria present makes it possible to choose an appropriate treatment. And with that the exacerbation of the symptoms, spread of the bacteria in the patient’s body, and the infection of others can be prevented.
Faster and more accurate
At present, if a patient comes to the doctor with symptoms of pneumonia then only bacterial cultures of lung mucus or a tissue biopsy can identify the pathogen responsible. These cultures are very time-consuming, whereas speed is vital to limit any possible damage to the lungs. ‘It costs at least 24 hours before the result is known’, says Michel Klerks. ‘Furthermore, some patients find it difficult to cough up the lung mucus because that requires a lot of strength. For this group in particular, for example weak patients or small children, our invention could have a major impact.’
Membrane filters bacteria out
In a NanoNextNl project, Innosieve Diagnostics and RadboudUMC developed the proof of concept to directly filter bacteria from the exhaled air. The most important part of the concept is a silicon nitride membrane with very uniform tiny holes. The holes easily allow air to pass through but retain the bacteria present. These can then be analysed directly. This is the first technology that can directly detect bacteria in exhaled air, including aerosols: small fluid drops in air´, says Klerks.
The most important component is a silicon nitride membrane with very uniform tiny holes.
The membrane easily allows air through but retains the bacteria (shown green here).
To demonstrate that this concept can be applied in clinical practice Innosieve, with the help of a Valorisation Grant from NanoNextNL, is now collaborating with Radboud UMC on elaborating the proof of concept and further validation studies. ‘A rapid, affordable and easy diagnostic method would mean a considerable improvement in many clinical settings. We have already demonstrated that the principal works.’ The entrepreneur hopes that within 7 to 10 years the device will be available in doctors’ consultation rooms as well.
Regaining control of your bladder
More than half of all women who have recently given birth suffer from unwanted urine loss during movements such as coughing, jumping or laughing. One fifth of these women still suffer from these symptoms a year later. In autumn 2015, start-up company LifeSense launched Carin, a system that helps women to regain control of their bladders within a few months.
‘Unwanted urine loss is often still a taboo subject’, says Valer Pop, business development manager at the Holst Centre in Eindhoven. ‘Only ten percent of women with this type of complaint seek medical advice, even though seventy percent of them can experience a complete recovery.’
To commend his product, Pop uses the example of Alicia, a sportive young mother of two children. After giving birth twice she suffers from unwanted urine loss. Ashamed of her symptoms she wears incontinence pads every day and does not practice any sports in busy places.
Sensor measures urine loss
Via the start-up LifeSense, Pop is now bringing a solution to the market, which bears the name Carin. Carin consists of a flexible sensor incorporated into a pantyliner that measures when and how much urine loss occurs. The sensor is linked to a personalised training programme put together by pelvic floor specialists. ‘The sensor very accurately measures urine loss and sends this information to the mobile phone of the woman concerned. An app on her phone keeps track of when and how much urine is lost and during which activity’, explains Pop.
Carin consists of a sensor, a pad and an app.
Personal training scheme
The woman subsequently receives a tailored and personalised training programme based on the incontinence profile. Because the problem lies in the pelvic floor muscles and that is where the solution is to be found as well. ‘These muscles are often damaged during childbirth’, says Pop. ‘By very specifically training these muscles the problem can be reduced or even completely overcome within several months. There are an awful lot of different exercises for these muscles that are aimed at different muscle groups. By analysing the measurements of the urine loss, Carin can precisely indicate for each woman which muscles need training and how.’
The biggest advantage of this approach is that it puts the woman in control, says Pop. ‘Many women stop prematurely with doing general exercises because they do not benefit enough from these.’ With Carin participants can choose whether they want to work with a physiotherapist who analyses the data from the app or with an e-coach who on the basis of the information provided by the pelvic floor specialists automatically puts together a personalised training programme.
Clinical trials are currently taking place with volunteers who want to test the system. The Pelvic Care Center of Maastricht University Hospital and the Archipel Zorg Groep are involved in these. Professionals such as pelvic floor physiotherapists, gynaecologists, nurses and urologists are following the trials. Via the Valorisation Programme of NanoNextNL the spin-off from the Holst Centre has received tips and recommendations for improving the business case. ‘Worldwide there are about 400 million women who could benefit from this innovation’, says Pop. ‘ This offers fantastic business opportunities with a large societal impact.’
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