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Magnetic immunoassay for detection of staphylococcal toxins in complex media Alexey V. Orlov, Julia A Khodakova, Maxim Petrovich Nikitin, Anna Olegovna Shepelyakovskaya, Fedor Aleksandrovich Brovko, Alexander G. Laman, Eugene V Grishin, and Petr I. Nikitin Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/ac303075b • Publication Date (Web): 17 Dec 2012 Downloaded from http://pubs.acs.org on December 17, 2012
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Analytical Chemistry
Magnetic immunoassay for detection of staphylococcal toxins in complex media Alexey V. Orlov†,‡ , Julia A. Khodakova†, Maxim P. Nikitin†,‡,§, Anna O. Shepelyakovskaya||, Fedor A. Brovko||, Alexander G. Laman||, Evgeny V. Grishin§, Petr I. Nikitin†,**†‡§|| A. M. Prokhorov General Physics Institute, Russian Academy of Sciences, 38 Vavilova St. Moscow 119991 Russia; Moscow Institute of Physics and Technology, Moscow, Russia; ShemyakinOvchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia; Pushchino Branch of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Pushchino, Moscow Region, Russia Method of highly sensitive registration of magnetic nanoparticles by their non-linear magnetization is used in a novel sandwich-type immunoassay for detection of staphylococcal toxins in complex media of virtually any volume, with increasing sensitivity at higher sample volume. The signal is read out from the entire volume of a non-transparent 3D fiber structure employed as a solid phase, which provides large reaction surface, quick reagent mixing as well as antigen immunofiltration directly in the course of the assay. The method has demonstrated near-linear dose-response curves within wide range of ~3 decades while detection of staphylococcal enterotoxin A (SEA) and toxic shock syndrome toxin (TSST) in neat milk without sample preparation. The limits of detection (LOD) as low as 4 and 10 pg/mL for TSST and SEA, respectively, were obtained in 2-hour format using 30-mL samples. The second, 25-minute format, showed the LOD of 0.1 and 0.3 ng/mL for the same toxins in a 150 µL sample. The developed immunoassay can be applied in food safety control, in vitro diagnostics and veterinary for a variety of research from express tests in the field to highly sensitive laboratory tests.
INTRODUCTION In recent decades, extensive research efforts have been focused on development of methods for fast detection of toxins produced by Staphylococcus aureus. Due to high virulence of these bacteria, their stability and increasing resistance to antibacterial medications, these toxins are widely present in the environment1. They are frequently responsible for diverse serious and potentially fatal illnesses such as severe gastrointestinal diseases and toxic shock2. Some toxins such as Staphyloccal enterotoxin B (SEB) potentially can be used as biological weapons3,4. One of the most common ways to contract staphylococcal enterotoxins (SEs) is through contaminated foods and water. Such foods do not exhibit any specific appearance, taste or odor5. *
To whom correspondence should be addressed. Phone/Fax: +74991350376. E-mail:
[email protected] † A.M. Prokhorov General Physics Institute, Russian Academy of Sciences, 38 Vavilova St. Moscow 119991 Russia ‡ Moscow Institute of Physics and Technology, Moscow, Russia § Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia || Pushchino Branch of Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow Region, Russia ACS Paragon Plus Environment
Analytical Chemistry
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Thermal processing such as pasteurization or heating kills only bacteria and does not affect SEs6. Therefore, the detection of staphylococcal enterotoxins in food should be done by immunological rather than microbiological methods, because the latter register bacteria only. Notably, the data on infective dose of SEs that produces symptoms of staphylococcal intoxication in humans are rather contradictory. Mossel et al.7 mentioned that 10-20 µg of SEs cause initial symptoms of poisoning in adults. Later, Martin et al.8 reported that ingestion of less than 1 µg of SEs may cause intoxication in sensitive humans. In 2000, an outbreak of staphylococcal food poisoning was recorded in Japan. More than 13000 persons were reported to be intoxicated by contaminated low-fat milk, the average dose of SEs being only 0.02 - 0.1 µg per capita9. Based on these facts, one can conclude that high sensitivity and speed of immunoassays for food analysis are among the major factors for reducing the risks of outbreaks of staphylococcal foodborne illnesses. Recent immunochromatographic tests with gold labels for detection of SEB10 take not more than 10 min, are inexpensive and have no special requirements to personnel. However, the sensitivity of such assays in complex media such as milk or serum without additional amplification is not high – about 10 ng/ml. The assay result is visually evaluated by an operator as "yes/no". Such subjective interpretation of the non-quantitative assays may result in false-negative or false-positive results. The most sensitive commercial assays for staphylococcal toxin detection such as ELISA11-14, visual immunoassay15, enzyme-linked fluorescent assay16 feature the limit of detection within 0.10.5 ng/mL and the assay time of 1.5-4 hours. The assay time does not include sample preparation required for all these methods. Among the advanced developments, evanescent wave fluorescence array biosensors17 successfully demonstrated simultaneous detection of several toxins in food matrices on the level of 0.5 ng/mL for 30 minutes. With fluorescent hydrogel biochips, seven staphylococcal enterotoxins were detected in 1:20 diluted milk on the level of 0.1 - 0.5 ng/mL at 17-hour assay time18. The cantilever sensors19 detect SEs on the level of tens pg/mL, but instability due to fluctuations of temperature, pH and ionic strength of solutions significantly restricts their employment for highthroughput screening of food or analyzing of "dirty" samples. Application of magnetic beads for SEB detection in complex matrix by fluidic force discrimination assays (FFD)20,21 on a flat biochip, which was optically readout, showed remarkable detection limits ∼1 pg/ml for sequential20 and ∼1 fg/ml for semi-homogenous21 assays. However, one should keep in mind the low slope of the signal dependence upon concentration in such assays: only 4-fold growth of signal (0.6 orders) was observed when SEB concentration increased by 10 orders of magnitude21 (i.e., 1.15-fold growth of signal per 10-fold increase of SEB concentration). 2 ACS Paragon Plus Environment
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Analytical Chemistry
According to the authors, “it would be difficult to distinguish changes in concentration
