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Monitoring the life of bacteria in microdroplets

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Trying to establish better ways to test the effect of drugs on bacteria has led the Institute of Physical Chemistry of the Polish Academy of Sciences to develop a new monitoring technique. From a Jan.  11, 2017 news item on Nanowerk,

So far, however, there has been no quick or accurate method of assessing the oxygen conditions in individual microdroplets. This key obstacle has been overcome at the Institute of Physical Chemistry of the Polish Academy of Sciences.

Not in rows of large industrial tanks, nor on shelves laden with test tubes and beakers. The future of chemistry and biology is barely visible to the eye: it’s hundreds and thousands of microdroplets, whizzing through thin tubules of microfluidic devices. The race is on to find technologies that will make it possible to carry out controlled chemical and biological experiments in microdroplets. At the Institute of Physical Chemistry of the Polish Academy of Sciences (IPC PAS) in Warsaw a method of remote, yet rapid and accurate assessment of oxygen consumption by micro-organisms living in individual microdroplets has been demonstrated for the first time.

“Devices for the cultivation of bacteria in microdroplets have the chance to revolutionize work on the development of new antibiotics and the study of mechanisms responsible for the acquisition of drug resistance by bacteria. In one small microfluidic system it is possible to accommodate several hundred or even several thousand microdroplets – and to carry out a different experiment in each of them, for example with different types of microorganisms and at different concentrations of antibiotic in each drop,” describes Prof. Piotr Garstecki (IPC PAS), then explains: “For such studies to be possible, one has to provide the bacteria with conditions for development for even a few weeks. Thus, knowledge about the flow of oxygen to the droplets and the rate of its consumption by the microorganisms becomes extremely important. In our latest system we demonstrate how to read this key information.”

A Jan. 11, 2017 IPC PAS press release on EurekAlert, which originated the  news item, describes the work in more detail,

The bioreactors of the future are water droplets with culture medium suspended in a carrier liquid with which they are immiscible (usually this is oil). In the channel of the microfluidic device each droplet is longer than it is wide and it almost completely fills its lumen; sizes matched in this manner ensure that the drops do not swop places in the channel and throughout the duration of the experiment they can be identified without any problems. At the same time, there has to be a thin layer of oil maintained continuously between each microdroplet and the wall of the channel. Without this, the bacteria would be in direct contact with the walls of the channel so they would be able to settle on them and move from drop to drop. Unfortunately, when the microdroplet is stationary, with time it pushes out the oil separating it from the walls, laying it open to contamination. For this reason the drops must be kept in constant motion – even for weeks.

Growing bacteria need culture medium, and waste products need to be removed from their environment at an appropriate rate. Information about the bacterial oxygen consumption in individual droplets is therefore crucial to the operation of microbioreactors.

“It is immediately obvious where the problem lies. In each of the hundreds of moving droplets measurements need to be carried out at a frequency corresponding to the frequency of division of the bacteria or more, in practice at least once every 15 minutes. In addition, the measurement cannot cause any interference in the microdroplets,” says PhD student Michal Horka (IPC PAS), a co-author of the publication in the journal Analytical Chemistry.

Help was at hand for the Warsaw researchers from chemists from the Austrian Institute of Analytical Chemistry and Food Chemistry at the Graz University of Technology. They provided polymer nanoparticles with a phosphorescent dye, which after excitation emit light for longer the higher the concentration of oxygen in the surrounding solution (the nanoparticles underwent tests at the IPC PAS on bacteria in order to determine their possible toxicity – none was found).

Research on monitoring oxygen consumption in the droplets commenced with the preparation of an aqueous solution with the bacteria, the culture medium and a suitable quantity of nanoparticles. The mixture was injected into the microfluidic system constructed of tubing with Teflon connectors with correspondingly shaped channels. The first module formed droplets with a volume of approx. 4 microlitres, which were directed to the incubation tube wound on a spool. In the middle of its length there was another module, with detectors for measuring oxygen and absorbance.

“In the incubation part in one phase of the cycle the droplets flowed in one direction, in the second – in another, electronically controlled by means of suitable solenoid valves. All this looks seemingly simple enough, but in practice one of the biggest challenges was to ensure a smooth transition between the detection module and the tubing, so that bacterial contamination did not occur at the connections,” explains PhD student Horka.

During their passage through the detection module the droplets flowed under an optical sensor which measured the so-called optical density, which is the standard parameter used to evaluate the number of cells (the more bacteria in the droplets, the less light passes through them). In turn, the measurement of the duration of the phosphorescence of the nanoparticles, evaluating the concentration of oxygen in the microdroplets, was carried out using the Piccolo2 optical detector, provided by the Austrian group. This detector, which looks like a big pen drive, was connected directly to the USB port on the control computer. Comparing information from both sensors, IPC PAS researchers showed that the microfluidic device they had constructed made it possible to regularly and quickly monitor the metabolic activity of bacteria in the individual microdroplets.

“We carried out our tests both with bacteria floating in water singly – this is how the common Escherichia coli bacteria behave – as well as with those having a tendency to stick together in clumps – as is the case for tuberculosis bacilli or others belonging to the same family including Mycobacterium smegmatis which we studied. Evaluation of the rate of oxygen consumption by both species of microorganisms proved to be not only possible, but also reliable,” stresses PhD student Artur Ruszczak (IPC PAS).

The results of the research, funded by the European ERC Starting Grant (Polish side) and the Maria Sklodowska-Curie grant (Austrian side) are an important step in the process of building fully functional microfluidic devices for conducting biological experiments lasting many weeks. A system for culturing bacteria in microdroplets was developed at the IPC PAS a few years ago, however it was constructed on a polycarbonate plate. The maximum dimensions of the plate did not exceed 10 cm, which greatly limited the number of droplets; in addition, as a result of interaction with the polycarbonate, after four days the channels were contaminated with bacteria. Devices of Teflon modules and tubing would not have these disadvantages, and would be suitable for practical applications.

Here’s a link to and a citation for the paper,

Lifetime of Phosphorescence from Nanoparticles Yields Accurate Measurement of Concentration of Oxygen in Microdroplets, Allowing One To Monitor the Metabolism of Bacteria by Michał Horka, Shiwen Sun, Artur Ruszczak, Piotr Garstecki, and Torsten Mayr. Anal. Chem., 2016, 88 (24), pp 12006–12012 DOI: 10.1021/acs.analchem.6b03758 Publication Date (Web): November 23, 2016

Copyright © 2016 American Chemical Society

This paper is behind a paywall.

Polish researchers develop Superman’s kryptonite?

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It’s not precisely kryptonite but rather a krypton-oxygen compound according to a March 2, 2016 news item on ScienceDaily,

Theoretical chemists have found how to synthesize the first binary compound of krypton and oxygen: a krypton oxide. It turns out that this exotic substance can be produced under extremely high pressure, and its production is quite within the capabilities of today’s laboratories.

A March 2, 2016 Institute of Physical Chemistry of the Polish Academy of Sciences (IPC PAS) press release (also on EurekAlert), which originated the news item, provides more information about Superman’s kryptonite, real krypton, and the new synthesized compound,

Crystals of kryptonite, a material deadly to Superman and his race, were supposed to have been created within the planet Krypton, and therefore most likely under very high pressure. The progenitor of the name, real krypton, is an element with an atomic number of 36, a noble gas considered to be incapable of forming stable chemical compounds. However, a publication in the journal Scientific Reports by a two-man team of theoretical chemists from the Institute of Physical Chemistry of the Polish Academy of Sciences (IPC PAS) in Warsaw, Poland, presents the possibility of synthesizing a new crystalline material in which atoms of krypton would be chemically bonded to another element.

“The substance we are predicting is a compound of krypton with not nitrogen, but oxygen. In the convention of the comic book it should, therefore, be called not so much kryptonite as kryptoxide. So if Superman’s reading this, he can stay calm – at the moment there’s no cause for panic!” laughs Dr. Patrick Zaleski-Ejgierd (IPC PAS) and adds: “Our krypton monoxide, KrO, probably does not exist in nature. According to current knowledge, deep in the interiors of planets, that is, the only place where there is sufficient pressure for its synthesis, oxygen does not exist, nor even more so, does krypton.”

Compounds of krypton have been produced before, in the laboratory under cryogenic conditions. They were, however, only single, linear and small molecules of the hydrogen-carbon-krypton-carbon-hydrogen type. The Polish chemists wondered if there were conditions in which krypton would not only bond chemically with another element, but also in which it would be capable of forming an extensive and stable crystal lattice. Their search, funded by an OPUS grant from the Polish National Science Centre, involved the researchers using genetic algorithms and models built on the so-called density functional theory. In the field of solid state physics, this theory has for years been a basic tool for the description and study of the world of chemical molecules.

“Our computer simulations suggest that crystals of krypton monoxide will be formed at a pressure in the range of 3 to 5 million atmospheres. This is a huge pressure, but it can be achieved even in today’s laboratories, by skillfully squeezing samples in diamond anvils,” says PhD student Pawe? Lata (IPC PAS).

Crystal lattices are built from atoms or molecules arranged in space in an orderly manner. The smallest repetitive fragment of such structures – the basic ‘building block’ – is called a unit cell. In crystals of table salt the unit cell has the shape of a cube, the sodium and chlorine atoms, arranged alternately, are mounted on each corner, close enough to each other that they are bound by covalent (chemical) bonds.

The unit cell of krypton monoxide is cuboid with a diamond base, with krypton atoms at the corners. In addition, in the middle of the two opposite side walls, there is one atom of krypton.

“Where is the oxygen? On the side walls of the unit cell, where there are five atoms of krypton, they are arranged like the dots on a dice showing the number five. Single atoms of oxygen are located between the krypton atoms, but only along the diagonal – and only along one! Thus, on each wall with five krypton atoms there are only two atoms of oxygen. Not only that, the oxygen is not exactly on the diagonal: one of the atoms is slightly offset from it in one direction and the other atom in the other direction,” describes Lata.

In such an idiosyncratic unit cell, each atom of oxygen is chemically bound to the two nearest adjacent atoms of krypton. Zigzag chains of Kr/O\Kr\O/Kr will therefore pass through the crystal of krypton monoxide, forming long polymer structures. Calculations indicate that crystals of this type of krypton monoxide should have the characteristics of a semiconductor. One can assume that they will be dark, and their transparency will not be great.

Theorists from the IPC PAS have also found a second, slightly less stable compound of krypton: the tetroxide KrO4. This material, which probably has properties typical of a metal, has a simpler crystalline structure and could be formed at a pressure exceeding 3.4 million atmospheres.

After formation, the two kinds of krypton oxide crystals could probably exist at a somewhat lower pressure than that required for their formation. The pressure on earth, however, is so low that on our planet these crystals would undergo degradation immediately.

“Reactions occurring at extremely high pressure are almost unknown, very, very exotic chemistry. We call it ‘Chemistry on the Edge'”. Often the pressures needed to perform syntheses are so gigantic that at present there is no point in trying to produce them in laboratories. In those cases even methods of theoretic description fail! But what is most interesting here is the non-intuitiveness. From the very first to the last step of synthesis you never know what’s going to happen,” says Dr. Zaleski-Ejgierd – and he returns to his computer where simulations of subsequent syntheses are nearing their end.

I don’t usually include images of the researchers but these guys dressed up for the occasion,

Chemists from the Institute of Physical Chemistry of the Polish Academy of Sciences in Warsaw have found a method of synthesizing a new crystalline material in which atoms of krypton would be chemically bonded to another element. (Source: IPC PAS, Grzegorz Krzy¿ewski) Metodê syntezy pierwszego trwa³ego zwi¹zku kryptonu znale¿li chemicy-teoretycy z Instytutu Chemii Fizycznej PAN w Warszawie. (ród³o: IChF PAN, Grzegorz Krzy¿ewski)

Chemists from the Institute of Physical Chemistry of the Polish Academy of Sciences in Warsaw have found a method of synthesizing a new crystalline material in which atoms of krypton would be chemically bonded to another element. (Source: IPC PAS, Grzegorz Krzy¿ewski)
Metodê syntezy pierwszego trwa³ego zwi¹zku kryptonu znale¿li chemicy-teoretycy z Instytutu Chemii Fizycznej PAN w Warszawie. (ród³o: IChF PAN, Grzegorz Krzy¿ewski)

Here’s a link to and a citation for the paper,

Krypton oxides under pressure by  Patryk Zaleski-Ejgierd & Pawel M. Lata. Scientific Reports 6, Article number: 18938 (2016) doi:10.1038/srep18938 Published online: 02 February 2016

This paper is open access.

Chiral breathing at the Institute of Physical Chemistry of the Polish Academy of Sciences (IPC PAS)

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An April 17, 2014 news item on ScienceDaily highlights some research about a polymer that has some special properties,

Electrically controlled glasses with continuously adjustable transparency, new polarisation filters, and even chemosensors capable of detecting single molecules of specific chemicals could be fabricated thanks to a new polymer unprecedentedly combining optical and electrical properties.

An international team of chemists from Italy, Germany, and Poland developed a polymer with unique optical and electric properties. Components of this polymer change their spatial configuration depending on the electric potential applied. In turn, the polarisation of transmitted light is affected. The material can be used, for instance, in polarisation filters and window glasses with continuously adjustable transparency. Due to its mechanical properties, the polymer is also perfectly suitable for fabrication of chemical sensors for selective detection and determination of optically active (chiral) forms of an analyte.

The research findings of the international team headed by Prof. Francesco Sannicolo from the Universita degli Studi di Milano were recently published in Angewandte Chemie International Edition.

“Until now, to give polymers chiral properties, chiral pendants were attached to the polymer backbone. In such designs the polymer was used as a scaffold only. Our polymer is exceptional, with chirality inherent to it, and with no pending groups. The polymer is both a scaffold and an optically active chiral structure. Moreover, the polymer conducts electricity,” comments Prof. Włodzimierz Kutner from the Institute of Physical Chemistry of the Polish Academy of Sciences (IPC PAS) in Warsaw, one of the initiators of the research.

An April 17, 2014 IPC PAS news release (also on EurrekAlert), which originated the news item, describes chirality and the breathing metaphor with regard to this new polymer,

Chirality can be best explained by referring to mirror reflection. If two varieties of the same object look like their mutual mirror images, they differ in chirality. Human hands provide perhaps the most universal example of chirality, and the difference between the left and right hand becomes obvious if we try to place a left-handed glove on a right hand. The same difference as between the left and right hand is between two chiral molecules with identical chemical composition. Each of them shows different optical properties, and differently rotates plane-polarised light. In such a case, chemists refer to one chemical compound existing as two optical isomers called enantiomers.

The polymer presented by Prof. Sannicolo’s team was developed on the basis of thiophene, an organic compound composed of a five-member aromatic ring containing a sulphur atom. Thiophene polymerisation gives rise to a chemically stable polymer of high conductivity. The basic component of the new polymer – its monomer – is made of a dimer with two halves each made of two thiophene rings and one thianaphthene unit. The halves are connected at a single point and can partially be rotated with respect to each other by applying electric potential. Depending on the orientation of the halves, the new polymer either assumes or looses chirality. This behaviour is fully reversible and resembles a breathing system, whereas the “chiral breathing” is controlled by an external electric potential.

The development of a new polymer was initiated thanks to the research on molecular imprinting pursued at the Institute of Physical Chemistry of the PAS. The research resulted, for instance, in the development of polymers used as recognising units (receptors) in chemosensors, capable of selective capturing of molecules of various analytes, for instance nicotine, and also melamine, an ill-reputed chemical detrimental to human health, used as an additive to falsify protein content in milk and dairy products produced in China.

Generally, molecular imprinting consists in creating template-shaped cavities in polymer matrices with molecules of interest used first as cavity templates. Subsequently these templates are washed out from the polymer. As a result, the polymer contains traps with a shape and size matching those of molecules of the removed template. To be used as a receptor in chemosensor to recognize analyte molecules similar to templates or templates themselves, the polymer imprinted with these cavities must show a sufficient mechanical strength.

“Three-dimensional networks we attempted to build at the IPC PAS using existing two-dimensional thiophene derivatives just collapsed after the template molecules were removed. That’s why we asked for assistance our Italian partners, specialising in the synthesis of thiophene derivatives. The problem was to design and synthesise a three-dimensional thiophene derivative that would allow us for cross-linking of our polymers in three dimensions. The thiophene derivative synthesised in Milan has a stable three-dimensional structure, and the controllable chiral properties of the new polymer obtained after the derivative was polymerised, turned out a nice surprise for all of us”, explains Prof. Kutner.

Here’s a link to and a citation for the paper,

Potential-Driven Chirality Manifestations and Impressive Enantioselectivity by Inherently Chiral Electroactive Organic Films by  Prof. Francesco Sannicolò1, Serena Arnaboldi, Prof. Tiziana Benincori, Dr. Valentina Bonometti, Dr. Roberto Cirilli, Prof. Lothar Dunsch, Prof. Włodzimierz Kutner, Prof. Giovanna Longhi, Prof. Patrizia R. Mussini, Dr. Monica Panigati, Prof. Marco Pierini, and Dr. Simona Rizzo. Angewandte Chemie International Edition Volume 53, Issue 10, pages 2623–2627, March 3, 2014. Article first published online: 5 FEB 2014 DOI: 10.1002/anie.201309585

© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

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