From a lab on a chip to a lab on a fiber

Currently, laboratory tests require very expensive equipment and reagents that need to be stored under strictly defined conditions, as well as the work of highly qualified specialists. Therefore, behind the apparent simplicity of the procedure of blood sampling for tests, there are large financial and time costs. These costs are one of the main reasons for the increase in the cost of medical services worldwide.

Moreover, in agricultural regions and developing countries, this situation is complicated by the fact that patients have to get to the clinic within a few hours or even days. In such conditions, they often do not return to collect the results, and many of them simply cannot afford to diagnose such life-threatening diseases as malaria or tuberculosis.

The only way to overcome these problems is to improve laboratory diagnostic tools. They should be cheap and portable enough to be used in any conditions. They should be so easy to use that nurses, hospice staff, soldiers and even patients themselves can conduct testing with minimal training. And finally, the new generation of laboratory instruments should provide fast and accurate results, which will save patients from waiting and excitement.

The fulfillment of all these requirements is by no means science fiction. The key to achieving the desired is the use of optical fiber, which is practically no different from those that have enveloped the whole world with a network that provides continuous high-speed exchange of a huge amount of information.

The new technology is called "laboratory on fiber". In addition to being an affordable alternative to traditional laboratory technologies, it provides previously unavailable capabilities. For example, such fibers can be placed inside industrial equipment to ensure product quality control and leak detection. They can be used to monitor water quality in canals and purification systems, to study the composition of ocean water and as indicators to identify chemical threats. In the future, perhaps in a few decades, they can be injected into the human body to monitor the dynamics of the disease or the metabolism of drugs.

Diagnostics with the help of lightFor the functioning of the laboratory on the fiber, light waves of the near infrared range are used, which makes it possible to determine the concentrations of chemical compounds and biological molecules in solution with high accuracy.

The figure schematically shows the principle of operation of the device.

• The laser transmits light energy to the fiber CORE.
• The INCLINED GRATING reflects light waves with certain (resonant) wavelength values from the fiber core.
• When TARGET MOLECULES bind to receptors on the fiber surface, a wavelength shift and a change in the resonance intensity occur.
• The GOLDEN MIRROR on the end of the fiber reflects the light waves stored in the core in the opposite direction – to the spectrometer located on the opposite end of the fiber.
• The SPECTROMETER (not shown in the image) registers changes in the returned light, which makes it possible to estimate the concentration of target molecules.

For the first time, researchers thought about creating low-cost mobile laboratories in the late 1960s. At that time, engineers learned how to place thousands (eventually billions) of transistors on a single board the size of a fingernail, which marked the beginning of the development of powerful microprocessors, as well as high–speed high-capacity storage devices. With the improvement of microelectromechanical systems, they are increasingly being used in biomedical research to create compact microchips, which are many sensors placed on a single miniature silicon oxide board.

The design and complexity of such systems, known as a lab on a chip, vary widely. A typical kit includes miniature pumps and valves that direct the movement of a small sample of liquid, for example, a drop of blood, through microchannels to different registration zones. In these zones, target molecules contained in the blood, such as glucose or antibodies to certain viruses, react with compounds deposited on the surface of the chip, which changes the voltage on the electrodes or the current strength in the conductor. The chip amplifies these signals, digitizes and analyzes them, after which they are displayed on a portable display by wires or using radio waves. The whole process takes no more than 20 minutes.

Sensors created on the principle of "laboratory on a chip" technology are ideal for use in remote clinics or directly at the patient's bedside. However, their widespread use for other tasks is hindered by completely insurmountable circumstances. For example, in a humid environment (inside the body or outdoors), the metal conductors of the chips are eroded or a short circuit occurs in them, which makes it impossible for the sensors to function correctly. In addition, many chips are manufactured using substances that are toxic to humans, such as arsenic. However, their biggest disadvantage is their size. Modern batteries, processors and transmitters allow you to create devices with a size of at least a few square centimeters, which does not allow them to be inserted into blood vessels.

To overcome these problems, researchers are looking for ways to replace electrical circuits with optical ones. The use of light waves instead of electric current to register chemical reactions allows photonic chips to work in aqueous solutions, makes them immune to electromagnetic radiation, provides the possibility of their use in a wide temperature range, and minimizes risks to biological tissues.

Light waves have another important advantage. Electronic devices transmit information using a single quantifiable parameter – current or voltage. Optical devices encode data in a similar way by changing the intensity or amplitude of light waves. In addition, light radiation can be divided into many components with different wavelengths, which makes it possible to use many data transmission channels. This significantly increases the information capacity of the circuit, several times increasing the sensitivity of photonic laboratories on a chip compared to electronic ones.

However, despite many promising qualities, the creation of a "laboratory on fiber" is still associated with several serious difficulties. Just like electronic chips, optical systems are too large to be introduced into the body. In this respect, they are even inferior to electronic chips, since light waves cannot be "compressed" to sizes smaller than the wavelength. Therefore, photonic chips using near-infrared light, the wavelength of which is about 1 micrometer, are much larger than their electronic counterparts, consisting of crystals smaller than 30 nanometers.

The manufacture of sensors for photonic laboratories on a chip is also associated with large financial costs, since it requires the manufacture of complex systems of lenses and mirrors that would form light rays and direct light waves to chemical registration zones. Silicon-based photonic integrated circuits, currently used in the production of data transmission equipment, cost several hundred dollars each. At the same time, an electronic chip can be purchased literally for a penny.

What will overcome these shortcomings and make full use of the wonderful properties of light waves? A promising option is to replace photonic chips with fiberglass.

Since fiberglass became a cheap, affordable material in the 1980s, researchers have been experimenting with various methods of creating sensors for a fiber-based laboratory. Currently, several groups have already proposed their concepts of combining materials and assembly methods for cheap large-scale production of stable systems.

For example, scientists from Carleton University in Ottawa (Canada) together with colleagues from the University of Mons (Belgium) and Jinan University (China) are working on the creation of a fiber laboratory, which, despite the simplicity of production, provides extremely accurate results.

The manufacturing process involves the use of very cheap standard fiber used in telecommunications equipment. These fibers, as thick as a human hair, have a core and an outer layer covered with a protective polymer shell. While the outer layer consists of pure silicon oxide, the core has germanium oxide inclusions that increase the refractive index – a parameter indicating the speed of light transmission in the material. When light waves moving through the core hit the outer shell at a certain angle, a small difference between the refractive coefficients of the two layers ensures that the light beam returns back to the core. Due to this phenomenon, known as total internal reflection, light waves can travel along the fiber core for many kilometers along a trajectory resembling the trajectory of a ball in table tennis. The ability to move light waves over long distances with minimal losses will allow the use of fiber-based laboratories to maintain the functioning of systems submerged in ocean waters, or serving many clinics at the same time.

Where to place the probe?One of the best approaches to creating a laboratory on fiber is to place a chemical detector or probe on the outer surface of an optical fiber (see the illustration "Diagnostics with light").

However, this is not the only option. The two most promising alternatives are illustrated below.

At the endGold nanostructures placed on the end of a standard optical fiber reflect light waves back to the detector located at the opposite end.

When target molecules attach to chemical receptors on a given coating, they increase its thickness, which changes the properties of the reflected light. This approach was developed by researchers at the University of Sannio, Italy.Inside the fiber

This principle implies the use of a "microstructured fiber" directing a light beam inside an air tunnel surrounding a hollow or solid glass core.

After the introduction of a gaseous or liquid sample into the core or other cavities, its chemical properties are determined by analyzing the spectral profile of light reaching the end of the fiber. The approach was developed by researchers of the Institute of Light Science. Max Planck, Germany and the Technical University of Denmark.To transform an inert fiber into a chemical sensor, it is necessary to choose the principle of placement of the registration zone.

Some groups are considering the option of placing the probe on the end of the fiber or inside an air tunnel in the outer layer of experimental-type fibers known as microstructured fiber (see the inset "Where to place the probe?"). However, Canadian researchers have come to the conclusion that the simplest and, accordingly, the cheapest is to use a segment of the outer surface of the fiber with a length of 1-10 mm.

A chemical compound is applied to this segment that reacts with target molecules, such as enzymes contained in the blood or, for example, biologically active substances. There are many different compounds that can be used as such reagents, as well as many methods of applying them to the fiber surface. One of the most promising approaches is implemented in two stages. First, a thin layer of metal is applied to the surface using one of the standard methods (spraying, thermal vacuum evaporation or deposition by chemical reduction). After that, the probe is immersed in a salt bath containing a solution of aptamers – short synthetic DNA chains specifically binding to the target molecule, which are attached to the coating applied to the surface.

Chemical companies can synthesize a huge number of aptamers that react with various molecules, including proteins, toxins and even compounds that are part of the shell of living bacteria. This makes it possible to create fiber-optic probes to detect almost any chemical or biological substance. The used probe can be cleaned from the original aptamers by chemical washing and prepared for reuse by applying the necessary new aptamers.

After the fiber has acquired the ability to bind target molecules, it remains only to learn how to count them. Light is used for this. A miniature light source, such as a laser diode, is attached to one end of the fiber, naturally directing light through the fiber core to a chemical probe. The opposite end of the fiber is covered with a golden mirror, which allows analyzing the returned waves using a conventional spectrometer.

However, these reflected waves will say nothing about changes occurring on the probe surface, such as the attachment of target molecules to aptamers. In order to register these changes, the light must be able to interact with the outer surface of the probe, on which the aptamers are applied. Therefore, it is necessary to somehow force the light to leave the core of the fiber.

Many earlier solutions to the problem involved removing part of the outer layer of the fiber by polishing or chemical engraving, which would ensure that the light fibers would come out. However, such impacts reduce the strength of the fiber. Moreover, their execution requires high accuracy, which significantly complicates and increases the cost of production.

Canadian researchers have proposed a solution consisting in creating a so-called inclined lattice in the core of the fiber. In fact, it is a permanent hologram created by exposing the fiber to a powerful ultraviolet laser for several minutes before applying a metal coating and reagent. To do this, a plastic buffer is first dissolved around the probe area, which makes it transparent to ultraviolet light. The laser beam passes through a phase mask, which is a glass plate covered with grooves, which divides it into two beams. The fiber is placed behind a phase mask, where light waves overlap each other, forming a so-called interference zone, in which intervals of high radiation intensity are interspersed with intervals of no radiation. This leads to the breaking of certain molecular bonds in the fiber core material, increasing its scattering coefficient in the zones corresponding to the intervals of high radiation intensity. As a result, a so-called lattice is formed, consisting of zones with a higher reflection coefficient.

These zones are shaped like disks and act as imperfect mirrors, each of which reflects a small amount of light moving inside the probe. When they are positioned perpendicular to the fiber, the light is reflected in the opposite direction and returns to its source. In order for the reflected light to be redirected to the fiber surface, during laser processing, the fiber is positioned at an angle, which ensures the formation of an inclined lattice.

An important point is that the reflecting grid does not redirect the entire light beam from the core. It reflects only light radiation with resonant wavelength values determined by the distance between the reflecting disks and the refractive properties of the fiber. When resonant light waves leave the core, they are reflected from the surface of the fiber and pass back through its inner layer. In such conditions, they overcome only a few centimeters, after which they are completely absorbed by the outer plastic shell of the fiber. Therefore, by analyzing the spectrum of light waves returned by the golden mirror to the spectrometer, it is possible to identify the missing resonant wavelengths and estimate the loss of radiation intensity.

When the probe is placed in the analyzed solution, for example human blood, the target molecules bind to the aptamers on its surface, changing its ability to reflect and absorb light. Such a small physical transformation changes the value of the resonant wavelength. Subsequent analysis of the characteristics and severity of these changes makes it possible to determine the concentration of molecules reacting with the probe coating.

The sensitivity of such a device depends on the material used to attach the aptamers binding to the target molecules to the probe surface. The metal coating can increase the sensitivity of the laboratory on the fiber by 4 orders of magnitude.

This is explained by rather strange laws of physics. The thickness of the coating, as a rule, varies in the range of 10-100 nanometers, which corresponds to about 1/100 of the wavelength of the resonant radiation reaching it. When making a coating from any other material, most of these light waves would pass through it without reacting in any way to changes occurring with the surface coating, such as the attachment of target molecules to aptamers.

Critical coverage. Nanofragments of metallic sputtering (brown) on the surface of the fiber probe form "hot spots" of electromagnetic energy (yellow). This layer increases the sensitivity of the laboratory on the fiber by 4 orders of magnitude.However, the metal coating has unique properties that cause light waves to interact with it.

The metal is a conductor, the electrons of its surface layer oscillate in the electromagnetic field generated by the light wave. Under certain conditions provided by the wavelength, the angle of incidence of light and the properties of the surface material, electron oscillations in metal coating nanoparticles resonate with electromagnetic field oscillations. Such electron resonances are called localized surface plasmons. Just as the air inside a guitar amplifies the sound of vibrating strings, they absorb the energy of an electromagnetic field-inducing light wave, creating hot spots of electromagnetic energy that can spread several hundred nanometers beyond the metal surface. Plasmons are much larger than the nanoparticles around which they form, which significantly increases the likelihood that the light wave will react to molecular processes occurring on the surface of the probe.

In one of the experiments, the researchers demonstrated that glass fiber, which does not have a metal coating, makes it possible to register biotin (aka vitamin H, vitamin B7, coenzyme R) at a concentration of 20 micrograms per liter of test solution. At the same time, the gold-coated probe reduces the minimum recorded concentration of this vitamin to 2 nanograms per liter. This roughly corresponds to the concentration obtained by dissolving a pinch of table salt in a 25-meter swimming pool. (Gold was used to cover the probe for reasons of its inertia and safety for the body.)

Thanks to its miniaturization and exceptionally high sensitivity, fiber-based laboratory technology can be used to perform a variety of tasks. For example, in 2012, researchers at the Catholic University of Leuven, Belgium published a paper in which they used gold-plated probes to identify the smallest possible variations in DNA fragments. The results obtained by them indicate that in the future this inexpensive tool may provide the possibility of rapid and accurate genetic screening for such complex diseases as cystic fibrosis, various types of cancer and certain infections.

Illuminating life: A fiber lab was used to register the growth of living skin cells (purple). The photo was taken using a fluorescent microscope.More recently, Canadian researchers from Carleton University have demonstrated that a fiber lab can be used to monitor the vital activity of cells.

As part of this experiment, they immersed the probe in a culture of skin cells, some of which adhered to its surface. Upon receipt of nutrients, the cells divided, increasing the density of the surface layer of the probe, which changed the profile of the light radiation coming to the spectrometer. And, on the contrary, under the influence of toxins, cells died and exfoliated from the surface of the probe, which reduced the density of its surface layer and manifested itself by other changes in the spectrogram. Such sensors can be used to study living tissues, since they are so small that they cannot affect the behavior of cells.

The ultimate goal of the Canadian researchers' work is to create a fiber-based laboratory that can be injected into the human body to monitor biological changes occurring in it in real time. They are already planning experiments aimed at evaluating the possibility of using a fiber-optic probe to detect metastatic cells in the bloodstream. The first stage of this work will be carried out in a laboratory, and the second – on animal models. Scientists hope in the truest sense of the word to shed light on the process by which malignant cells penetrate into healthy organs. They also hope that their efforts will help in the development of new methods of screening for cancer pathology, less invasive than traditional approaches such as biopsy. For example, a doctor will insert a fiber-optic probe into a blood vessel using a disposable syringe needle. This procedure will be no more painful than a normal injection into a vein.

Most likely, the appearance of laboratories on a chip on the commercial market will not happen earlier than in five years. One of the difficulties that the developers have yet to overcome is to increase the stability of the surface coating of the probe to ensure that it can be stored for several months without losing the ability to bind to target molecules.

In any case, fiber-based laboratory technology in many areas of biomedicine is already very close to competing in economic efficiency and quality of results with existing diagnostic methods. The first swallow may well be a blood test. Imagine that you take a diagnostic kit, inject yourself into the pad of your finger and squeeze out a drop of blood onto a system of fiber-optic probes. Within a few minutes, the device automatically sends the results by e-mail to your attending physician, who will contact you within a few hours and help solve the problem.

Article by Yanina Shevchenko et al. Surface plasmon resonance fiber sensor for real-time and label-free monitoring of cellular behavior is published in the journal Biosensors and Bioelectronics.

Evgeniya Ryabtseva
Portal "Eternal youth" http://vechnayamolodost.ru 

31.03.2014