17 April 2015

Diagnostics using a smartphone: details

A smartphone attachment for molecular diagnostics has been created

Yulia Kondratenko, "Elements"

Our smartphones are more powerful than the computers that were used to launch the Apollo missions. But, what is even more remarkable, their possibilities are wider. Special attachments have already been created that allow smartphones to be used for microscopy and even for the diagnosis of eye diseases. And now American scientists have developed a nozzle and a program for a smartphone to turn it into a kind of mini-laboratory for ultrafast molecular diagnostics.

In biology, there are methods that allow you to obtain very important data (for example, allowing you to diagnose a patient), but at the same time simple in nature. One of these methods is molecular diagnostics using antibodies, using their ability to recognize certain types of molecules well. Antibodies are provided with tags, thanks to which they are easy to see (for example, fluorescent molecules are sewn to them). Then such antibodies are added to the sample, wait until binding occurs, and wash off the excess antibodies. As a result, by the fluorescence of the antibodies bound to the sample, it is possible to see where the molecules of interest to us are located.

A modification of this method is catching target molecules from a sample using antibodies as bait. To do this, relatively large balls are used, plastered with antibodies to what we are interested in. The balls need to be added to the sample, wait until their targets stick to the antibodies, and then remove the balls, and with them the target molecules.

Scientists managed to adapt a similar method for ultra-fast diagnostics, which can now be carried out using a conventional smartphone. To turn a smartphone into a diagnostic laboratory, the device will need to be equipped with a special nozzle, which has a sample compartment and a light source, as well as an application that sends data for analysis to the server, and then demonstrates the results of a molecular study to the user (Fig. 1).

Fig. 1. The scheme of the molecular diagnostics system using a smartphone. A – diagnostic scheme: first, you need to prepare a sample of the tissue under study (for example, blood); then, using antibodies, mark the targets with special balls; then remove the diffraction pattern and send it to the server for analysis. B – the device of the nozzle. C – interaction of the smartphone and the server. Here and below are images from the discussed article in PNAS

With the help of such a device, it will be possible to check the presence of certain molecules on the surface of the sample cells (for example, blood). It will be necessary to add balls with antibodies to the molecules of interest to the cells (such balls can be bought, as well as any other laboratory reagents). The smartphone camera is used to capture the diffraction pattern that is formed when the rays are scattered on a mixture of balls and the cells under study. The image is sent to the server, which restores the location of cells and balls from the diffraction pattern (Fig. 2). The more balls stuck to a certain cell, the more target molecules on its surface.

Fig. 2. An example of the operation of the diagnostic system. The upper row – the balls did not attach to the cage, the lower row – the balls stuck to the cage (the size of the balls is 7 microns). Diffraction patterns alone are not enough for correct recognition, the algorithm also uses data on the transparency (Transmittance) and phase shift (Phase) of objects – according to these parameters, the balls strongly contrasted with the studied cells.

Now we know a lot of molecules that are markers of malignant cells, and if we use balls with antibodies to such molecules, we can determine whether there are malignant cells in the sample. You can also identify any other cells with specific molecules on the surface (for example, this way you can find different types of cells of the immune system). For analysis, several types of balls carrying different antibodies can be used simultaneously and cells with a certain set of markers can be found in such an experiment. In this case, it is necessary to use balls of different sizes, or made of different materials, so that balls with different types of antibodies can be distinguished from each other.

Scientists have made sure that the ratios of the balls binding to cells coincide with the ratios of gene expression of the target molecules – that is, using a smartphone, it will be possible to conduct not only qualitative, but also quantitative molecular analysis. To test their system in practice, the researchers used it to analyze tissue samples from 25 patients with cervical cancer. For control, the samples were also examined by traditional histological methods. The samples were divided into categories depending on the severity of the disease (high risk, low risk, benign tumor), and for two methods – classical and new – the characteristics of the samples coincided. At the same time, the analysis using the new smartphone system not only did not require expensive equipment and complex manipulations, but also gave results very quickly – the entire procedure, including sample preparation and data analysis, takes 45 minutes. Another advantage of the new system is a large viewing angle. Tens of thousands of cells are placed in one visual field of a smartphone camera, and they can be analyzed simultaneously. When diagnosing using microscopy, much smaller numbers of cells can be studied simultaneously.

Scientists have also figured out how their system can be used for DNA analysis. To check whether there is a certain DNA in the sample (for example, virus DNA), you need to use two types of beads with attached single-stranded DNA fragments complementary to the target sequence. First, the target DNA is fished out of the sample using balls of the same type. Then balls of a different type are added to them. If there were molecules in the sample with ends corresponding to sequences on two different beads, then at the output we will get dimers of beads connected by molecules of trapped DNA. Such dimers form a characteristic diffraction pattern, and if we see it, it means there is target DNA in the sample (Fig. 3). This method was able to detect attomoles (10-18 moles) of target DNA, and this without PCR amplification. This sensitivity is comparable to the sensitivity of the most accurate methods of DNA research (theoretically, using PCR, you can detect a single copy of DNA in a sample, in practice, the sensitivity of the method is one or two orders of magnitude lower - that is, 10-22-10-21 moles of DNA).

Fig. 3. Search for the target DNA in the sample. Two types of beads with attached single-stranded DNA fragments are used, for which the target molecule can be pulled from the sample. The dimers of the beads that have caught the target molecule give a characteristic diffraction pattern by which the presence of the target DNA molecules in the sample can be determined.

The authors of the new molecular diagnostics system believe that since smartphones are spreading so fast, they need to come up with as many useful applications as possible. Of course, to use their system, special reagents and certain laboratory skills are needed, so it is unlikely that anyone will be able to do molecular analyses in the near future. Nevertheless, the new diagnostic system is probably one of the simplest, fastest, cheapest and at the same time working systems imaginable. So it will surely find its use.

Source: H. Im et al. Digital diffraction analysis enables low-cost molecular diagnostics on a smartphone // PNAS.

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