New spin on NMR
13
C
solenoid. The design was evalTypically requiring milligram quantities uated with natural-abundance of material, 13C NMR has never been known for its sensitivity. In this issue of and 13C-enriched Analytical Chemistry (p. 2424), Andrew samples at a Webb and Raju Subramanian of the Uni- magnetic field versity of Illinois at Urbana-Champaign of 11.7 T. Natural-abundance describe how the use of solenoidal mi13 C NMR of sucrocoils for high-resolution 13C NMR crose had a 3c can substantially improve the limits of limit of detecdetection. tion (LOD) of "Our question was: How far can we 18 ug (52 nmol) The 13C direct detection probe. (A) The 13C-ZH double resonance push microcoil technology?" says Subramanian, a postdoctoral research asso- with an acquisicircuit. (B) The 1H decouple circuit. ciate. "You're dramatically reducing the tion time of 90 min. As would be expected even less volume from a conventional probe." A traction. The 3a LOD for sucrosefroma 13 C-enriched material is required. The 3CT 1-D 13C decoupled HMQC spectrum typical conventional NMR probe has at 13 375 ng (4 2 nmol) with [3- Clleast 275 uL of observed volume, which LOD was 1.6 ug (4.5 nmol) for a 14-min acwas reduced to 1 uL in the miniaturized L-alanine with an acquisition time of less quisition time. probe. "There's a dramatic loss in terms than 30 s The potentially greatest use of miof number of spins. Can we partially crocoil 13C NMR will be with massThe "inverse detection" probe consists compensate for that by using more sen- of a solenoid tuned to the SH frequency and limited samples. With a total sample of sitive and efficient coils?" he asks. 100 ug, direct detection 13C NMR is an outer coil tuned to the 13C frequency, 13 possible with a "reasonable" acquisition for C decoupling. Because the probe deThe answer is yes, but it required time of around an hour. With 10 ug, tects 'H, the S/N is higher, but the chalcareful redesign of the electrical circuitry. The direct detection microprobe lenge is in suppressing the signal from pro- indirect detection is possible in a comtons bound to 12C rather than 13C. Suppres- parabletime.For 13C-enriched samples consists of a solenoid tuned to the 13C sion is accomplished with a pulse sequence the quantity required could be as little and deuterium frequencies, the former (known as heteronuclear multiple-quantum as 1 ug. This improvement in sensitivity to perform the experiments and the latcould make the need for milligram coherence) that enhances the signal from ter to shim and lock the sample. A 13 C-attached protons while the signal from quantities a distant memory. larger coil tuned to the *H frequency, the 12C-attached protons cancels by subfor proton decoupling, surrounds the Celia Henry
Weighing up TaySachs disease A biosensor for Tay-Sachs (T-S) disease based on a sensitive quartz crystal microbalance (QCM) could soon be available, according to Israeli scientists. T-S is a genetic disorder (affecting mainly people of Jewish descent) that results in a lack of the regulatory enzyme hexosaminidase A (hex A) that breaks down neuronal lipids. Symptoms usually appear in infants between four and six months when an otherwise healthy child will stop smiling, rolling, and crawling. Blindness and paralysis follow, and death occurs by about age five. A genetic test is available for putative carriers but it is expensive and timeconsuming, and only gives a statistical likelihood of a couple having a T-S baby. A blood test for the rapid detection of T-S
carriers and for detecting the disease early in pregnancy is much sought after, according to Itamar Willner of the Hebrew University of Jerusalem. Willner and his colleagues are developing such a technique for detecting the disease based on recognition of the defective oligonucleotide strand in the T-S gene. In principle, says Willner, the test could be carried out using saliva or a pinprick of blood. As afirststep, he and his team have devised a QCM device with an interface for the T-S oligonucleotide. The team applied a monolayer of oligonucleotide strands complementary to the mutation to a gold surface (on the quartz crystal of 9-MHz natural frequency) using thiol bridges. The process of detection then involves the coiling together of the T-S oligonucleotide with its complement in the QCM monolayer. This results in a concentrationdependentfrequencydrop in the QCM
crystal as the mass of the T-S strand is added by binding. In controls, the researchers found that the frequency of the bare gold-quartz crystal was not affected by the presence of the T-S oligonucleotide. They also found that noncomplementary oligonucleotides failed to give a frequency change with the coated device, demonstrating its specificity. (Chem. Commun. 1998,
Hanging in the balance'. A schematic of QCM detection of T-S genetic mutation. (Adapted with permission. Copyright 1998 Royal Society of Chemistry.)
Analytical Chemistry News & Features, July 1, 1998 4 4 3 A
News
839-40). "The problem of specificity is usually of major concern in DNA sensors," Willner told Analytical Chemistry. "Our research shows that a 7-base mutation of DNA is easily recognized and differentiated from the target DNA analyte. Thus, ,i seems that our sensor exhibits and reveals high specificity." To increase sensitivity of their device the team devised a way to amplify the microgravimetric signal using antibody technology. Beforehand, they generated antibodies to the double strand of oligonucleotide and by adding these to the sample solution they could boost the frequency change to about 8 Hz as the antibody complexes to the bound T-S strand. The next step in development is to find a way to regenerate the surface monolayer, which will lead to a viable device. Willner adds, "The method of organizing the sensing interface, as well as the QCM transductton, could be applied for the analysis of any other genetic disorder." David Bradley
Biosensor detects estrogen - mimicking compounds A new fiber-optic biosensor for detecting the presence of estrogenic compounds in aqueous samples has been developed at IA in Ann Arbor, MI. The sensor—which wiil be marketed byThreeFold Sensors, a subsidiary of IA— was conceived by John Erb-Downward and brought to fruition by a collaboration between Judith L Erb and co-workers at IA and James L Wittliff and Wolfgang Raffelsberger at the University of Louisville Medical School. According to Judith Erb, the biosensor, referred to as the Endotect system, obtains results in 20 min or less. .n addition to determining whether a compound is ble of binding to an estrogen receptor, the also determines whether a compound is an agonist or antagonist for cific receptor she says The system is portable and offers a quick-screening method for the detection of endocrine disrupters an important class of comDounds believed to cause reproductive and ohvsical abnormalities in wildlife and possiblv in humans The Endotect system relies on binding measurements between a known concentration of fluorophore-labeled estrogen receptor and a chemically sensitized optical fiber. An evanescent field is generated on the surface of the fiber, causing the estrogen receptor to fluoresce. Unbound fluo444 A
rescent receptors exhibit minimal excitation because the evanescent field only extends about 1000 A from the surface of the fiber, says Erb. Receptor binding can therefore be measured without having to separate the bound from unbound receptor she says A sample suspected of containing an estrogenic compound is mixed with the fluorophorelabeled receptor and Endotect system for the detection of estrogenic compounds. introduced into the system bv means of a disposable sensor with compounds that we knew had some cartridge Upon sample introduction sort of estrogenic activity; however, we changes in binding between the receptor have not yet tried it in a real world sample," molecule and the fiber are recorded says Erb. Interferences could occur if something destroyed the receptor, she "The receptor molecule is a recombisays. "It probably wouldn't be great to use nant human receptor, which has been exit on samples coming right out of a pipeline pressed in yeast," says Erb. Therefore only where you might have high concentrations compounds that bind to the human estroof strong acids that would degrade the progen receptor are expected to have a positeins. I visualize two places where it'd be tive response, she adds. very useful," she comments. "The first case Two different sensor fibers are used so is where you know what the compound is that agonists can be distinguished from and you want to determine if it has estroantagonists, says Erb. "Type 1 fibers congen-mimicking capabilities. The other OT16* tain a weak estrogen on their surface, and is where you want to determine if type 2 fibers possess a nucleotide that rething in an environmental sample has posembles the binding region of the nuclear estrogen response element." If a compound tential estrogen activity" decreases binding to a type 1 fiber and inBritt Erickson creases binding to a type 2 fiber, it is likely to be an estrogenic agonist. On the other hand, if a compound decreases binding to a Analyzing phosphine type 1 fiber and either decreases or has no effect on binding to a type 2 fiber, it is at mud flats likely to be an estrogenic antagonist. Although phosphine (PH3) was detected as Injection of the sample mixture into a early as 1927 in sewage purification plants, type 1 fiber will indicate whether the samchemists ignored the investigations, even ple contains a compound capable of affecthushed and fought them. ing estrogen-mediated signal transduction; "Today we know that besides the [inhowever, the system is not intended to dustrial] sources for phosphine, natural identify the compound. "This sensor is dephosphate reduction [also] occurs," says signed for preliminary screening in the Gtinther Gassmann of the Biological Instienvironment," says Erb. "Its response will tute Helgoland (Germany). tell you if there is anything in the sample Natural phosphate reduction was bethat has estrogen-mimicking capabilities. If lieved to be impossible until recently. Gassthe answer is yes, then you start trying to mann was the first in Germany to develop a track down what is causing the positive reliable device to quantitatively measure response," she explains. phosphine in river and marine sediments. His apparatus is a mobiie double-cryotrapping The biosensor exhibits a response to a device with a phosphorus-selective thermivariety of natural and synthetic estrogenic onic detector. Mobility is important for compounds at levels of physiological and him—several times a year he cruises off the environmental interest. "We've tested it
Analytical Chemistry News & Features, July 1, 1998
