Review pubs.acs.org/ac
Biosensors as 21st Century Technology for Detecting Genetically Modified Organisms in Food and Feed Mary A. Arugula, Yuanyuan Zhang, and Alex L. Simonian*
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Department of Materials Engineering, Auburn University, Auburn, Alabama 36849, United States
CONTENTS
Electrophoretic and PCR Methods for GMO Detection GMO Biosensors Optical Biosensors Piezoelectric Biosensors Electrochemical Biosensors Conclusions Author Information Corresponding Author Notes Biographies Acknowledgments References
and an expression terminator (T), by inserting foreign DNA, which enables the expression of an additional protein conferring new characteristics, for example, herbicide tolerance, resistance to virus, antibiotic, and insect resistance. There are two particular sequences inserted into most transgenic plants, promoter of the 35S subunit of rRNA of the cauliflower mosaic virus (CaMV35S) and the terminator of nopaline synthase gene (TNOS) from Agrobacterium tumefaciens. These are used widely in commercial production of transgenic vegetables under the brand names such as soy Roundup Ready, the maize MaisGard, and the tomato Flavr Savr.4 Currently, the global status of commercialized GM crops reached 170 million hectares in a total of 29 countries, as revealed by ISAAA 2011(Figure 2). Among them the U.S. remains the top with 69 million hectares raising maize, soybean, cotton, canola, sugar beet, alpha-alpha, papaya, and squash, followed by Brazil and Argentina.5 Despite the great progress of technology, these modified foods have not gained worldwide acceptance in the general public because of raised consumer concerns, environmental issues, transparent regulatory oversight, and skepticism in government bureaucracies. During the early development of this field, when pesticides and other tolerant crops were introduced, it was thought to be safe and harmless for consumers. However, only over a decade, this technology has shown its true harmful implications which now have led to an ongoing debate on increasing research efforts evaluating the risks associated with the introduction of GMO into agriculture (e.g., potential gene flow to other organisms, agricultural diversity destruction, allerginicity, resistance to antibiotics, and gastrointestinal problems).6 Additionally, economical and moral issues with realization of contamination of non-GMOs with GMOs came into play. Therefore, several countries, including EU countries, Japan, Australia, New Zealand, Thailand, and China have implemented mandatory labeling for bioengineered foods. In the EU, strict restrictions were imposed on the import and introduction of legislation requiring mandatory food labeling in cases where more than 0.9% of the food ingredients (considered individually) are of GMO origin. However, the U.S. legislation instead opted for voluntary labeling and requested companies for U.S. Food and FDA approval before their launch into market.7,8 Consequently, 90% of the consumers have no idea what has been quietly introduced into their daily based food consumption and what impact they might cause in the near future.
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he history of genetically modified organisms (GMOs) can be traced to the year 1971, when Ananda M. Chakrabarthy discovered a multiplasmid hydrocarbon degrading bacteria Pseudomonas putida that was capable of digesting an oil spill 2 orders of magnitude faster than four similar strains.1 Since then, little more than 2 decades, this landmark research paved the way for a “biotech revolution” that allowed genetic transformation of virtually all terrains of life on earth. Mainly in the agricultural sector, in the years between 1997 and 1999 as much as 70−80 million acres were quickly converted to raise genetically modified (GM) food and crops.2,3 Predominantly, >40% of the corn, >50% of the cotton, and >45% of soybean acres of land and at least 2/3rds of all the U.S. processed foods contained GMOs. What caused this dramatic revolution lies in the fact that GMOs are unique, and they were mankind-created by forceful modification of their genome through gene technology. Genetic transformation/modification occurs by alteration of an organism gene cassette (Figure 1) consisting of an expression promoter (P), a structural gene (“encoding region”),
Figure 1. (a) Schematic representation of gene cassettes, consisting of a promoter (P), a structural gene (“coding region”) and a terminator (T); (b) frequently, two (or more) cassettes are transferred together and integrated into the host genome (horizontally bars) at one or several sites. © XXXX American Chemical Society
Special Issue: Fundamental and Applied Reviews in Analytical Chemistry 2014 Received: September 11, 2013
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dx.doi.org/10.1021/ac402898j | Anal. Chem. XXXX, XXX, XXX−XXX
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Figure 2. Global status of GM crops revealed by ISAAA 2011. Reprinted with permission from the ISAAA Webpage. Copyright 2011 ISAAA.
Table 1. Summary of the Detection Method, Target Gene/Sequence, Detection Limit, And Linear Range for Selected GMO Biosensors in the Review methods
target sequence/gene
detection limit
SPR SPR SPR SPR (a) BIACORE (b) SPREETA ECL-DNA SERS MS and SERS QCM QCM DPV ASV LSV EIS SWV
P35S and T-NOS 35S from soy, maize 35S from maize and ApoE genes from human blood GM Maize
1 nM 2.5 nM 2.5 nM and 50 nM (a) 2.5 nM (b) 10 nM 5 nmol/L 0.1 pg/mL 11 nM 0.25 ng/mL 4.7 × 105 copies 1 nM/L 4.38 × 10−12 mol/L 3 × 102 copies/reaction 4 pM 0.6% of transgenes
CaMV35S genes (cry1A(b) and cry1A(c)) in Bt rice 35S sequence of Bt-176 maize pflp gene in tobacco EPSPS gene in RR soybean P35S CaMV35S CBH 351 from maize CaMV35S cryIa/b and MON810 in maize flour
linearity range
ref
0.001−2.5 μM 2.5−25 nM 0−25 nM and 0−250 nM
13 16 17 18
5 nmol/L−5 μmol/L 1.0 pg/mL−10 ng/mL 25−100 nM 0.25−1.0 ng/mL
21 23 24 31 29 32 36 39 40 41
0−120 nM/L 1.2 × 10−11−4.8 × 10−8 mol/L 0−3 × 105 copies/reaction 0−269 pM 0.5−0.9%
target genes and sequences, limit of detection, and linear range.
Lack of transparent information hampers the public’s right to choose and freedom to make a choice on their consumables. The GMO database (http://www.gmo-compass.org/eng/gmo/db/) shows most of the processed foods available in the grocery stores, for example bread, baked foods, dairy products, egg products, chocolates, meat, and beverages contain GMO ingredients or additives prepared from soy and maize. Despite crucial concerns, the global cultivation of genetically modified crops is constantly expanding which drives one to demand to establish different ways to sort food and feed that consist or contain genetically modified organisms. Therefore, it has become important for the development of reliable and rapid methods of GMO detection, identification, tracing and quantification GMO analysis. Although there are numerous analytical methods for detection of GMOs, only a limited number of commercialized products may be found.9 The present Review will focus on biosensors as a cutting edge technology based on optical, electrochemical, piezoelectric transducers for GMO detection (screening) and quantification and summary (Table 1) of the detection methods used,
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ELECTROPHORETIC AND PCR METHODS FOR GMO DETECTION Numerous analytical methods have been developed for reliable determination of the presence or absence of genetically modified organisms in various food products. Most of these methods rely on two strategies either based on detection of inserted foreign DNA or detection of the novel protein that is specifically expressed in transgenic plants. Protein based testing methods include one-dimensional SDS gel electrophoresis, Western blot, immunochemical assays (ELISA), and lateral flow strip methods which are simple and specific but are not suitable for processed foods due to loss of epitopes during processing. Additionally, gel electrophoresis and ethidium bromide staining are laborious, time-consuming, and toxic. DNA based testing methods include polymerase chain reaction (PCR), ligase chain reaction (LPR), nucleic acid sequence based amplification (NASBA), fingerprinting techniques B
dx.doi.org/10.1021/ac402898j | Anal. Chem. XXXX, XXX, XXX−XXX
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very sensitive changes in the reflectivity of the gold surface. These changes can indicate the extent of binding of the analyte such as target DNA to the immobilized ligand (probe), which can be observed by the increase in mass and the refractive index.14,15 Mariotti et al. first reported their application of a SPR-based biosensor for screening analyses of GMOs. Target sequences P35S and T-NOS that many GMOs have in common were detected. Synthesized 25-mer oligonucleotides were first immobilized onto 11-mercaptoundecanol and a carboxylated dextran modified gold sensor chip. Hybridization at sensor surface between 25-mer P35S and T-NOS (concentration ranging from 0.001−2.5 μM) and their fully complementary oligonucleotide probes were monitored. The detection limit was 1 nM for both P35S and T-NOS with a CV
