Grant for NIR work to help athletes

Scientists from the Department of Biological Sciences at the University of Essex have received a grant of £28,571 from the UK’s Engineering and Physical Sciences Research Council to help in their studies of the use of near infrared (NIR) spectroscopy to determine the levels of oxygen in muscle. This helps athletes to optimise their training schedules and racing strategies. The funding will help design and develop a lightweight, portable device that can be worn comfortably in training and feed information wirelessly in real time to the coach. This will allow optimal targeting of training sessions in the field.

Professor Chris Cooper, Professor Ralph Beneke and Dr Caroline Angus of Essex’s Medical Optics Group will be working with physicists, engineers and computer scientists at University College London who will design the instrumentation. The Essex team will be carrying out testing and optimisation.

Professor Cooper explains that “exercise uses up oxygen and therefore how much oxygen is in the muscle is a measure of whether the oxygen being delivered is keeping up with its consumption. The key to the project is to take this data from the scientists to the coaches so they can use it to help optimise the way athletes warm up, or to design pacing strategies telling athletes when it’s the right time for them to speed up or conserve energy during a race.”

The team aims to have a working prototype in trials by spring 2008. For more information see www.essex.ac.uk/bs/mog and an article by Caroline Angus in NIR news last year (www.impublications.com/nir/abstract/N17_0721).

Earlier work with an NIR probe on the vastus lateralis muscle during a cycling test with an athlete. Reproduced with permission from C. Angus, NIR news 17(7), 21 (2006).

Reactions observed in a single cell

Bioengineers at the University of California, Berkeley, have discovered a technique that for the first time enables the detection of biomolecules’ dynamic reactions in a single living cell. By taking advantage of the absorptions of organic and inorganic molecules, the team of researchers, led by Luke Lee can determine in real time whether specific enzymes are activated or particular genes are expressed, all with unprecedented resolution within a single living cell.

The technique, described in the 18 November 2007 issue of Nature Methods (doi: 10.1038/nmeth1133), could lead to a new era in molecular imaging with implications for cell-based drug discovery and biomedical diagnostics. The researchers point out that other techniques, such as nuclear magnetic resonance, can at best provide information about a cluster of cells. But to determine the earliest signs of disease progression or of stem cell proliferation, it’s necessary to drill down deeper to the molecular dynamics within a single cell.

“Until now, there has been no non-invasive method that exists that can capture the chemical fingerprints of molecules with nanoscale spatial resolution within a single living cell,” said Lee. “There is great hope that stem cells can one day be used to treat diseases, but one of the biggest challenges in this field is understanding exactly how individual cells differentiate. What is happening inside a stem cell as it develops into a heart muscle instead of a tooth or a strand of hair? To find out, we need to look at the telltale chemical signals involved as proteins and genes function together within a cell.”

The researchers tackled this challenge by improving upon conventional optical absorption spectroscopy. “For conventional optical absorption spectroscopy to work, a relatively high concentration of biomolecules and a large volume of solution is needed in order to detect these subtle changes in frequencies and absorption peaks,” said Lee. “That’s because optical absorption signals from a single biomolecule are very weak, so you need to kill hundreds to millions of cells to fish out enough of the target molecule for detection.”

Light scatters from metallic nanoplasmonic particles upon excitation by an external light source. The metallic nanoparticles were coupled with biomolecules which gave much stronger absorptions and enabled reactions to be followed within a single living cell. (Graphic by Gang Logan Liu and Luke Lee/UC Berkeley, USA.)

The researchers came up with a novel solution to this problem by coupling biomolecules, the protein cytochrome c in this study, with tiny particles of gold measuring 20–30 nm long. The electrons on the surface of metal particles such as gold and silver are known to oscillate at specific frequencies in response to light, a phenomenon known as plasmon resonance. The resonant frequencies of the gold nanoparticles are much easier to detect than the weak optical signals of cytochrome c, giving the researchers an easier target.

Gold nanoparticles were chosen because they have a plasmon resonance wavelength ranging from 530 nm to 580 nm, corresponding to the absorption peak of cytochrome c.

“When the absorption peak of the biomolecule overlaps with the plasmon resonance frequency of the gold particle, you can see whether they are exchanging energy,” said study co-lead author Gang Logan Liu, who conducted the research as a UC Berkeley PhD student in bioengineering. “This energy transfer shows up as small dips, something we call ‘quenching,’ in the characteristic absorption peak of the gold particle.”

A relatively small concentration of the molecule is needed to create these quenching dips, so instead of a concentration of millions of molecules, researchers can get by with hundreds or even dozens of molecules. The sensitivity and selectivity of the quenching dips will improve the molecular diagnosis of diseases and be instrumental in the development of personalised medicine, the researchers said.

The researchers repeated the experiment matching the protein haemoglobin with silver nanoparticles and achieved similar results. “Our technique kills two birds with one stone,” Lee said. “We’re reducing the spatial resolution required to detect the molecule at the same time we’re able to obtain chemical information about molecules while they are in a living cell. In a way, these gold particles are like ‘nano-stars’ because they illuminate the inner life of a cellular galaxy.”

Other researchers on the UC Berkeley team are Yi-Tao Long, colead author and postdoctoral scholar in bioengineering; Yeonho Choi, a PhD student in mechanical engineering; and Taewook Kang, a postdoctoral scholar in bioengineering.

Doping detection

One of the most common substances used for doping is EPO (erythropoietin), which is difficult to detect. In an era when there are increasing numbers of “copies” of biotechnologically produced medications (biosimilars), it is also becoming more and more difficult to detect the difference between the body’s own EPO and that made biosynthetically. Chemists at Vienna University of Technology (TU) working jointly with ARC Seibersdorf, are developing a new analytical method, based on MALDI mass spectrometry, to track down the perpetrators of doping.

“With the aid of MALDI mass spectrometry, a method that is used for non-destructive desorption/ionisation of large molecules, especially biopolymers, we compare the deceptively similar ‘humanised’ form of EPO with the body’s own substance. The two samples differ in the structure of the amino acid chains and/or in that of the associated sugar chains. Depending on the structure of these sugar chains and where they bind to, we can recognise whether this is a natural or biosynthetic EPO”, explains Professor Günter Allmaier of the Institute of Chemical Technologies and Analytics at Vienna University of Technology.

Previous methods, for example iso electric focusing, exhibit several weaknesses. First, it takes between two and three days to obtain the test results. Furthermore, the method is regarded as difficult to automate, and is based on antibodies which can detect EPO in urine but sometimes are too nonspecific and do not distinguish the structure sufficiently precisely. Allmaier and his co-workers are concentrating now on a search for suitable analytical strategies that can detect recombinant EPO directly in urine. Lab-on-chip technology is to be combined with laser-based time-of-flight mass spectrometry. Following the testing phase, Allmaier estimates that the method may reach the patentable stage around 2009 and provide a valuable support in the fight against doping. Allmaier points out that “the most essential point in our strategy is that we are developing a method with which the EPO molecule itself is detected. All the other methods used so far have been indirect.”

EPO preparations increase the production of red blood cells, which in turn transport more oxygen in the blood. As a result, the organism’s performance improves. That is why EPO has been misused for doping as far back as the late 1980s, mainly in endurance sports such as cycling. Recently, Günter Allmaier received the John Beynon Prize Award 2007 for the most innovative publication in Rapid Communications in Mass Spectrometry for the period 2005 to 2006 (http://www3.interscience.wiley.com/cgi-bin/fulltext/114298803/ PDFSTART). This work was also the starting point for intensive cooperation with Dr Reichel of ARC Seibersdorf’s doping control laboratory.

SPR imaging

Using a new imaging technique based on surface plasmon resonance (SPR), a fast and accurate profile of autoantibodies present in the blood serum of rheumatic patients can be made. This profile can give valuable information about the progress of the disease. A unique feature of the SPR technique is that it directly tests on blood serum, without complex preprocessing. A special chip will enable many parallel tests. Scientists from the University of Twente and the Radboud University Nijmegen, both in The Netherlands, describe the new imaging technique in the Journal of the American Chemical Society (doi: 10.1021/ja075103x).

The scientists have run tests on the serum of 50 RA patients as well as a control group of 29 people. Direct testing on blood serum is unique: in other techniques fluorescent labels and preprocessing are necessary to visualise the relevant proteins. The diluted serum is led over a special gold coated microchip containing a large number of spots with a specific peptide coating. Whenever these peptides interact with autoantibodies present in the serum, this process can be monitored by Surface Plasmon Resonance Imaging (SPR). Using laser light, all gold spots are scanned: the reflection of light of the spots changes whenever there is a molecular interaction on the spot. At a certain angle of light, there is no reflection at all: this is the so-called SPR dip undergoing a shift caused by the interaction. The technique goes beyond proving that autoantibodies are present: the interaction between the protein and the antibody can be monitored real-time and without any labels.

Autoantibodies are manufactured by the immune system as a reaction on the citrullinated proteins playing a role in rheumatoid arthritis. On a single chip, several types of peptides can be placed, for rapid parallel screening. The next step is to investigate in what way the patient profiles help to monitor the progress of the disease. This could lead to more personalised treatment in the future. The applications are not limited to monitoring rheuma or other autoimmune diseases: SPR imaging can be used for monitoring a wide range of biomolecular interactions.

The research was led by Dr Richard Schasfoort of the BIOS Lab-on-a-chip group, part of the MESA+ Institute for Nanotechnology of the University of Twente. He has cooperated closely with the Biomolecular Chemistry group of Professor Ger Pruijn at the Radboud University Nijmegen. The research has been financed by the Dutch Technology Foundation (STW) within a project called “Proteomics on a chip for monitoring autoimmune diseases”.