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Microfluidic hydrogel scaffold for 3D cell culture
James J. Leary and William H. Ingham present an intuitive derivation of Einstein’s best-known equation, E = mc 2 . The paper is a formal treatment of material from a lecture in an undergraduate course on instrumental analysis. NAK WON CHOI
One of the challenges in creating 3D cell cultures is the need for microscale control over the soluble chemical environment and the delivery of molecules with spatial and temporal control. Abraham Stroock, Lawrence Bonassar, and colleagues at Cornell University have now met the challenge by directly embedding microfluidic channels in scaffolds made of a hydrogel. They used convective mass transfer to control the distributions and fluxes of solutes in the bulk 3D cell culture. The resulting gradients are useful for understanding biological phenomena and can be used to develop spatially heterogeneous tissues for tissue engineering. Stroock, Bonassar, and colleagues picked calcium alginate hydrogel as the scaffold material. The hydrogel was biocompatible, was mechanically stable enough to replicate microstructures and define discrete convective paths, had high diffusive permeability to small and large molecules, and could be processed under physiological conditions. Externally controlled pressure-driven flows through the microchannels delivered solutes to the cells. Solutes were exchanged in two steps: interfacial convective mass transfer between the flowing solutions and 5 mm the walls of the microchannels, and molecular diffusion between the walls and the bulk of the scaffold. For reactive solutes, diffusion–reaction processes defined gradients of Fluorescence micrographs of a microfluconcentration at steady state. For nonreacidic scaffold with 25 × 10 6 live cells/mL; tive solutes, the steady-state distributions the upper image is a top view, the lower were uniform when the sources and sinks image a cross-sectional view. A dye was were part of the scaffold. The investigators continuously delivered through the microsay that the experimental setup achieved ef- channels and allowed to diffuse into the ficient exchange of solutes with the bulk of tissue. The green zones indicate regions the scaffold and quantitative control of the where the tissue was well supplied by soluble environment around the cells. (Nat. fluid flow from the microfluidic network; Mater. 2007, DOI 10.1038/nmat2022) dark regions were starved of metabolites.
Getting to E = mc 2
The authors suggest that this derivation is relevant in instrumental analysis courses because conservation of energy, conservation of momentum, and alternative reference frames are important concepts in the design of instrumentation. In addition, understanding the derivation of this equation helps students practice their analytical thinking skills. Finally, the authors note that although this equation is very familiar, people (including chemists) often misinterpret it. (J. Chem. Ed. 2007, 84, 1651–1654)
The science behind Floyd Landis’s guilty verdict The popular press pounced on the juicy tales of sexual abuse and blackmail, but what stood on trial during the May 2007 Floyd Landis v. the United States Anti-Doping Agency (USADA) hearing were the analytical techniques and procedures used to determine whether an athlete has cheated by doping. The Landis camp accused Laboratoire National de Dépistage du Dopage (LNDD), the French laboratory that tested Landis’s
urine samples during the 2006 Tour de France, of scientific incompetence and not following the established procedures for the two analytical tests used to detect synthetic testosterone. USADA maintained that the protocols were followed correctly and that the discovery of exogenous testosterone metabolites in urine collected from Landis after his stunning Stage 17 win was indisputable.
In its verdict released on September 20, 2007, the three-member arbitration panel upheld a positive doping test for Landis in a 2:1 decision. However, it wasn’t a clear victory for USADA. The panel found flaws in the way the preliminary GC/MS screening test for synthetic testosterone was conducted. But they decided that the mistakes weren’t severe enough to throw into doubt the data from the independent and more reliable
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carbon isotope ratio MS (IRMS) test. The arbitrators said the GC/MS analysis, the results of which triggered the IRMS test on Landis’s urine, wasn’t valid because it wasn’t performed in accordance with the rules set up by the World Anti-Doping Agency (WADA). Had the results from the GC/MS test been thrown out when they were first reported, Landis’s sample never would have been analyzed by the IRMS test, which resulted in his doping conviction. But the arbitrators didn’t have to consider that—they only had to rule whether doping had occurred. They agreed that the results of the IRMS test did indeed show synthetic testosterone in Landis’s urine.
The T/E ratio test
The preliminary GC/MS screen for testosterone in urine is known as the T/E ratio test. T stands for testosterone; E is for epitestosterone, a natural, inactive isomer of testosterone. In most individuals, the T/E ratio is ~1:1. A T/E ratio of 4:1 may indicate the presence of synthetic testosterone. WADA has es_ 4:1 is the tablished that a T/E ratio of > threshold that triggers further testing of an athlete’s sample. Upon collection, each sample from an athlete is split into two vials, A and B, and sample A is tested first. (For more details on sample handling and testing for sports doping, see Anal. Chem. 2007, 79, 5522–5528.) The T/E test has two parts—a screening phase and a confirmation phase. T and E are identified by the main MS fragment ions produced from their respective trimethylsilyl derivatives in the screening phase. Once a chromatogram is produced, the T/E ratio is estimated on the basis of the peak area ratio. If the _ 4:1, then a GC/MS conT/E ratio is > firmation test is performed. Two new aliquots, one that is hydrolyzed and one that isn’t, are prepared for this test. The aliquot without hydrolysis measures “free” T and E to verify that the urine sample did not break down. LNDD performed the first screen8824
Floyd Landis is appealing the ruling that he doped with synthetic testosterone during the 2006 Tour de France.
ing test on Landis’s A sample during the summer of 2006 and reported a T/E ratio of 4.9:1. They then began the confirmation test, but the internal standard, methyltestosterone, was too weak, and the test was rejected. A second confirmation test was performed, and the result showed a T/E ratio of 11.4:1. Next, the lab tested the B sample by using only the confirmation method in triplicate. These three confirmation tests yielded T/E ratios of 10.9:1, 11:1, and 11.1:1. The Landis side alleged that LNDD didn’t properly identify T and E in the confirmation testing, because they analyzed only one diagnostic ion in both the A and B sample confirmation tests. For an adverse analytical finding (AAF), a single diagnostic ion is sufficient in the screening phase of the test, and the arbitration panel confirmed that LNDD did follow the procedure. However, for the subsequent confirmation test, WADA protocols demand that at least three diagnostic ions be acquired and monitored for each peak
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to ensure that there are no interferences that could affect the quantification, abundance, or size of the peaks. Although LNDD acquired three diagnostic ions, they only monitored one during the confirmation test. “The WADA rules are very clear about using three ions,” says Paul Scott of the Agency for Cycling Ethics, who served as a consultant to Landis’s defense team. “They didn’t do that, so that was kind of the end of it.” The panel declared the lab’s results to be noncompliant with the necessary procedures required to report an AAF for the T/E ratio test. But the panel went on to say that even though LNDD failed to follow the prescribed procedures for the T/E test, the IRMS analysis still applied because it can act as a stand-alone test for the determination of a synthetic testosterone AAF.
The IRMS test
Because the T/E tests yielded positive results, LNDD performed the IRMS test. IRMS measures the relative abundance of two stable isotopes of the same element—in the case of testosterone, 12C and 13C. The test for synthetic testosterone by IRMS was developed in the 1990s by Don Catlin, who is with the Anti-Doping Research Institute and is the former director of the University of California Los Angeles Olympic Analytical Laboratory (Anal. Chem. 2007, 79, 3963–3965). This test is based on the different isotopic signatures that endogenous and synthetic testosterones produce. Synthetic testosterone isn’t made from scratch. Pharmaceutical manufacturers carry out a partial synthesis in which they start with a precursor plant compound, typically from yams, and perform a few synthesis steps to convert it into testosterone. The plant-derived compound happens to have less 13C than human testosterone, and this disparity is what the antidoping test looks for. “You are what you eat, in a very simplistic way,” says Larry Bowers of USADA.
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To conduct the carbon isotope says Brenna. “If you see a pattern test for synthetic testosterone, of a small peak, big peak, small OH the testing lab injects the sampeak, and you go to the correH3C ple into a GC/IRMS (sometimes sponding area of the chromatoalso called a GC/C/IRMS— gram on the second technique C stands for combustion) instruand see a small peak, big peak, H3C ment, which separates compounds small peak, it gives you confidence and converts them into CO2 to that you are looking at the right measure their 13C/12C ratios. The thing.” lab compares the ratios of several Other points were disputed, steroids in the testosterone metabincluding the lab’s chain of cusO olism pathway (LNDD monitored tody, calibration of its instrument, androsterone, etiocholanolone, and the mathematical corrections and 5-- and 5--androstanediol) Testosterone produced by the human body and synthetic made to the data, but ultimately, with the ratios of control steroids testosterone, which is derived from plants, have differthe panel found the scientific evithat aren’t present in the pathway ent ratios of 13 C/ 12 C. dence to be sound. It concluded (in this case, 11-ketoetiocholanothat LNDD followed the required lone or 5--pregnandiol). The numbers peaks, and you’ll be able to positively protocols for the IRMS test and that are complicated, but to get an AAF, identify the peaks that you are claiming the AAF from the test should stand. the results must show a difference beare the various diols,” says Scott. tween the ratio of at least one of the But Landis’s side argued that LNDD Implications for future doping testosterone metabolites and the ratio didn’t use the same conditions with tests of a control nonmetabolite that is >3‰, both instruments and that thus the reOn October 10, Landis announced that the limit set by WADA. “The theory is tention times of analytes in the GC/MS he has appealed the arbitration ruling that you get an alteration in the [testos- and GC/IRMS didn’t exactly match and will take his argument to the Switterone] metabolite carbon isotope raup and couldn’t be correlated. “The zerland-based Court of Arbitration for tio value but not in the control, berelative retention times are completely Sport. It remains to be seen whether cause the control is not directly affected off, to the point where you don’t have a any new scientific evidence will be inby the use of [synthetic] testosterone,” good feel that you’ve properly identified troduced or whether a new panel of says Catlin, who served as a witness for your peaks,” says Scott. arbitrators will respond any differently USADA during the hearing. But experts on the USADA side to the complex scientific arguments. LNDD performed the IRMS test pointed out that the retention times “The issues here were very complicated on Landis’s A sample, which showed shouldn’t correspond exactly, because for anyone not well versed in isotope a >3‰ difference in 13C/12C ratio for the data come from two entirely difwork,” says Scott. “It’s not easy to two of the four testosterone metabolites ferent instruments. “The GC/MS reunderstand.” versus controls. The lab concluded that tention time is really equivalent to the The case has raised the question an AAF should be reported according time in which the peaks elute from the of whether, in their current state, the to the WADA protocol. It followed up GC column, whereas in a GC/IRMS analytical procedures and protocols in with an IRMS test on the B sample and experiment, the peaks elute from the sports drug testing can be properly folobtained the same results. GC column and then go through varilowed at all times so that no one can Landis’s side asserted that the French ous stages of combustion and drying dispute the results. “It’s really easy to lab was not actually analyzing the isoand so forth before they get to the mass play Monday-morning quarterback and tope ratios of the correct compounds. spectrometer,” says J. Thomas Brenna see an i that’s not dotted or a t that’s GC/IRMS cannot directly identify of Cornell University, who testified for not crossed, but that in no way underrelevant metabolites, so a separate GC/ USADA at the hearing. “So they simply mined the validity or the reliability of MS analysis (which is different from the cannot, and should not, correspond.” the work that was done by the French one performed for the T/E test) is reInstead, USADA argued, the lab’s lab,” says Travis Tygart, chief executive quired. Peaks are identified by GC/MS method of comparing the order and officer of USADA. “But we always look and then compared with peaks in the heights of peaks to a reference standard for ways to improve, and we’ll learn lesGC/IRMS chromatogram. “In theory, in the chromatograms was enough to sons from this case just like we do in if the GC/MS is set up properly and it’s positively identify the analytes. “Not every case that goes forward.” a done under the same conditions, you’ll only are the standards consistent, but —Rajendrani Mukhopadhyay get a perfect correlation between the the patterns of peaks were consistent,” and Jennifer Griffiths D E C e m b e r 1 , 2 0 0 7 / A n a l y t i ca l C h e m i s t r y
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