Chemical Education Today
Reports from Other Journals
Research Advances by Angela G. King
Bio-Bar-Codes Speed DNA Detection
More Information
In 1985, the introduction of the polyermase chain reaction (PCR) primer-mediated enzymatic amplification method revolutionized research and medical diagnostic testing. Yet PCR remains complex, expensive, and timeconsuming due to enzymatic amplification. Alternative DNA-detection assays—employing radioactive labels, fluorophore, nanostructure-based labels, and electrochemical tags—exist, but none have the level of sensitivity PCR affords. Researchers at Northwestern University have recently employed nanotechnology to develop a method of labeling disease markers present in blood with unique DNA tags they have dubbed “bio-bar-codes”. Scanning for the DNA tags then allows the quick identification of diseases ranging from Alzheimer’s to smallpox. The new method utilizes two-component oligonucleotide-modified gold nanoparticles (NPs) and single-component oligonucleotide-modified magnetic microparticles (MMPs). Amplified DNA can be detected using a chip-based detection method. Prostate specific antigen (PSA) can be detected at concentrations of 10⫺18 M with the bio-bar-code amplification (BCA) method. To demonstrate the merit of this approach, researchers use an oligonucleotide sequence target associated with anthrax lethal factor (LF), a bioterrorism and biowarfare agent. An MMP with an iron oxide core and amine-modified silane coating is prepared and functionalized by attaching alkanethiol oligonucleotides complementary to a 12-mer portion of the target sequence with a linker. A gold NP is then modified with two different types of oligonucleotides: one complementary to a region on the target sequence (different from the region complementary to the modified MMP) and one complementary to a bar-code sequence unique to the target sequence. The MMP probes are then added to a solution of singlestrand target DNA. Later, NP probes are added and allowed to hybridize for 90 minutes. Both unreacted MMPs and MMPs with target-linked NPs are pulled to the reaction vessel wall with a magnetic field, and solution components are washed away with a buffer. The magnetic field is removed, and the system is heated to release the bar-code DNA. The magnetic field is then re-applied to remove the MMPs from solution, and the bar-code DNA is then ready for detection. Assay results are analyzed with the chip-based scanometric DNA array detection method. This method detects DNA through amplification driven by oligonucleotidemodified gold NPs and NP-promoted reduction of Ag⫹. The entire assay requires only 3–4 hours for any concentration of target sequence.
1. Nam, J.-M.; Stoeva, S.; Mirkin, C. Bio-Bar-Code-Based DNA Detection with PCR-like Sensitivity. J. Am. Chem. Soc. 2004, 126, 5932–5933. 2. A general description of PCR is available in Weller, D. PCR Amplification of DNA. J. Chem. Educ. 1994, 71, 340. 3. A description of the scanometric method is available online at http://www.globaltechnoscan.com/13thSep.-19thSep/ dna_det.htm (accessed Jul 2004) 4. A brief description of the role of anthrax LF can be found in the August 2004 Research Advances column ( J. Chem. Educ. 2004, 81, 1086).
1386
Journal of Chemical Education
•
The DNA-BCA assay involves the preparation of nanoparticle and magnetic microparticle probes and a nanoparticle-based PCR-less DNA amplification scheme. Reprinted with permission from J. Am. Chem. Soc. 2004, 126, 5932. Copyright 2004 American Chemical Society.
Vol. 81 No. 10 October 2004
•
www.JCE.DivCHED.org
Chemical Education Today
Reports from Other Journals O
OH
OH
HO OCH3
OCH3
Curcumin, a promising treatment for cystic fibrosis isolated from turmeric, a spice from Curcuma longa.
Treating Cystic Fibrosis from the Spice Rack Cystic fibrosis (CF) is a common, chronic, and progressive genetic disease that causes mucus to be thick and sticky. Approximately 30,000 children and adults in the U.S. suffer from CF, and the average life span for people with CF is 32 years. The disease is caused by mutations in the gene for the cystic fibrosis transmembrane conductance regulator (CFTR) protein. Approximately 10 million individuals in the U.S. are unknowing symptom-free carriers of the defective gene, since a person must inherit two defective genes, one from each parent, to have CF. In secretory epithelia cells, CFTR protein forms chloride ion channels and allows salt and water to move to the airway surface. Ninety percent of people with cystic fibrosis carry a specific mutation known as ⌬F508 CFTR. This mutation results in a misfolded protein that can still function as an ion channel, but is retained in the endoplasmic reticulum (ER) and later degraded in the proteasome instead of moving to the cell surface. It is hypothesized that ⌬F508 CFTR protein is held in the ER due to interaction with calciumbinding proteins in the lumen. This is supported by the release of ⌬F508 CFTR from the ER upon incubation with calcium pump inhibitors. Once released, ⌬F508 CFTR protein resides and functions at the cell’s surface. Curcumin is a low-affinity sarcoplasmic/endoplasmic reticulum calcium (SERCA) pump inhibitor, and for this reason researchers at Yale investigated the efficacy of oral curcumin treatment in functionally redistributing ⌬F508 CFTR protein in CF-affected mice. The results of treatment were determined by measurement of the membrane potential difference across the nasal epithelia. Curcumin treatment decreased the measured potential. ⌬F508 CFTR knockout mice, who do not express CFTR protein, were also administered curcumin orally but showed no change in the abnormal membrane potential. Earlier results demonstrated that other SERCA pump inhibitors, structurally quite different from curcumin, have similar effects. Curcumin [(E,E)-1,7-bis(4-hydroxy-3-methoxyphenyl)1,6-heptadiene-3,5-one], isolated in 1910, was the first known diarylheptanoid and has many interesting pharmacological properties. Curcumin accounts for 1.5–2.0% by weight of turmeric (a spice prevalent in Thai and Indian cuisine), and gives turmeric its characteristic yellow color. Curcumin is structurally similar to isoflavinoids that are proposed to bind to CFTR protein and alter its channel properties. Thus curcumin may bind to and stabilize the tertiary struc1388
Journal of Chemical Education
•
ture of CFTR protein and allow it to evade the ER chaperones. Previous studies have shown that humans can tolerate large oral doses of curcumin, and the doses given in this study reflect those present in commercially available dietary supplements routinely consumed by individuals. Based on its oral availability and the studies with mice reported here, curcumin holds great promise in the treatment of CF. However, until dosage, safety, and possible benefits are investigated in a clinical trial, CF patients are discouraged from self-administering curcumin.
More Information 1. Egan, M.; Pearson, M.; Weiner, S.; Rajendran, V.; Rubin, D.; Glockner-Pagel, J.; Canny, S.; Du, K.; Lukacs, G.; Caplan, M. Curcumin, a Major Constituent of Turmeric, Corrects Cystic Fibrosis Defects. Science 2004, 304, 600–602. 2. A procedure for isolating curcumin in an undergraduate teaching lab is available. Anderson, A.; Mitchell, M.; Mohan, R. Isolation of Curcumin from Turmeric. J. Chem. Educ. 2000, 77, 359–360. 3. A description of the study and more information on curcumin treatment is provided by the Cystic Fibrosis Foundation at http://www.cff.org/research/files/CurcuminQAFinal.pdf (accessed Jul 2004)
Salmonella versus Salsa Salmonellosis is a food-borne illness that results from consuming viable cells from any member of the Salmonella genus, gram-negative rod-shaped bacilli. Salmonella are widely distributed in our food chain and often contaminate raw meats, eggs, unpasteurized milk, and dairy products. Other sources of Salmonella include contact with infected turtles, dogs, and cats. In the United States, there are more
Table 1. Antibacterial Activity (g/mL) of Selected Compounds Isolated from Fresh Coriander Leaves against Salmonella cholersesuis ATCC 3560 a MICb
Compound Decanal (2E)-decenal (2E)-dodecenal (2E)-undecenal dodecanal (2E)-tridecenal Nonanal (2E)-hexenal Geraniol Gentamycin
100 50 6.25 12.5 100 25 100 100 200 12.5
aData
MBCc 100 50 6.25 12.5 100 200 200 100 400 12.5
from J. Ag. Food Chem. 2004, 52, 3329–3332. Copyright ©2004 American Chemical Society. Used with permission.
bMIC: cMBC:
minimum inhibitory concentration minimum bacterial concentration
Vol. 81 No. 10 October 2004
•
www.JCE.DivCHED.org
Chemical Education Today
than 500 fatal cases of salmonellosis each year. Although any Salmonella can cause septicemia, the most frequent source of this problem is Salmonella cholersesuis ATCC 3560. Currently no anti-Salmonella agent for food is available, and a safe and effective method to fight food-borne illness is needed. Salsa juice containing tomatoes, onions, coriander (also known as cilantro), and green chilies inhibits the growth of some bacteria, including Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. Volatile compounds identified in fresh coriander leaves, Coriandrum sativum L., have been proposed as the active components in salsa juice. (2E )dodecenal, (2E )-decenal, and decanal account for 85% of the amount of identified components in this coriander. An international team from California and Mexico recently reported that phytochemicals from fresh leaves of C. sativum possess bactericidal activity against Salmonella. The phytochemicals were assayed against Salmonella cholersesuis ATCC 3560. Major volatile compounds from C. sativum were subjected to an antibacterial assay, time-kill study, and an enzyme assay. All of these aldehydes were effective against S. cholersesuis and their activity correlates with the chain length between the hydrophilic aldehyde group and the hydrophobic alkyl tail of the molecule. (2E )-dodecenal was the most
www.JCE.DivCHED.org
•
potent bactericide tested, demonstrating a minimum bactericidal concentration (MBC) of 6.25 g/mL and a minimum inhibitory concentration (MIC) of 6.25 g/mL. Both the MBC and MIC of (2E )-dodecenal are more potent than gentamycin. The time-kill assay indicates both that (2E )dodecenal kills quickly, most likely by disrupting the cell membrane, and that the compound will act on bacteria at any growth stage. It is worth noting that other phytochemicals in salsa juice may contribute to its antibacterial activity. These compounds may synergize or antagonize the activity of coriander-volatile compounds.
More Information 1. Kubo, I.; Fujita, K.-I.; Kubo, A.; Nihei, K.-I.; Ogura, T. Antibacterial Activity of Coriander Volatile Compounds against Salmonella choleraesuis. J. Ag. Food Chem. 2004, 52, 3329–3332. 2. More information on salmonellosis is provided by the Center for Disease Control at http://www.cdc.gov/ncidod/dbmd/ diseaseinfo/salmonellosis_g.htm (accessed Jul 2004).
Angela G. King is Senior Lecturer in Chemistry at Wake Forest University, P. O. Box 7486, Winston-Salem, NC 27109;
[email protected].
Vol. 81 No. 10 October 2004
•
Journal of Chemical Education
1389
