Biosensors for Detection of Genetically Modified Organisms in Food and Feed

Chapter 10

Biosensors for Detection of Genetically Modified Organisms in Food and Feed Mary A. Arugula, Alex L. Simonian Auburn University, Department of Materials Engineering, Auburn, AL, USA

INTRODUCTION Since the 1990s genetically modified organisms (GMOs) have paved the way for a biotech revolution that allowed genetic transformation of virtually all terrains of life. Genetic engineering has led to the discovery of GMOs in modern biotechnology for the study of gene function. Agricultural crops were the first to be introduced with GMOs, and placed in the market. From 1997 to 1999, as much as 70–80 million acres were quickly converted to grow GM food and crops. More than 40% of the corn, 50% of the cotton, and 45% of soybean are GM and at least two-thirds of all processed foods in the United States contained GMOs (Gasser and Fraley, 2011; Holst-Jensen, 2009). What are GMOs? These are organisms whose unique genetic material has been altered by insertion of a new gene or by deletion of an existing one. Genetic transformation/modification occurs by alteration of an organism gene cassette (Figure 1) consisting of an expression promoter, a structural gene (“encoding region”), and an expression terminator, by inserting foreign DNA which enables to express an additional protein conferring new characteristics, for example, herbicide tolerance or resistance to a virus, antibiotics, or insects. Most transgenic plants are developed by the insertion of two particular sequences: the promoter of the 35S subunit of ribosomal RNA of the cauliflower mosaic virus (CaMV 35S) and the terminator of nopaline synthase gene (T-NOS) from soil bacteria, Agrobacterium tumefaciens. Farmers around the world use the commercial production of these transgenic vegetables under brand names such as soy Roundup. Biotech crops including Roundup Ready (RR) soybean, MaisGard maize, and the Flavr Savr tomato (Hemmer, 1997) reached 170 million hectares in total of 29 countries, as revealed by The International Service for the Acquisition of Agribiotech Applications in 2011. Of these, the United States remains the top, with 69 million hectares raising maize, soybean, cotton, canola, sugar beet, alfalfa, papaya, and squash, followed by Brazil and Argentina (James, 2012). While GMOs are not pertained to only plant development, engineering of most transgenic animals began in the 1990s. Several types of GM animals include cattle, sheep, pigs, chicken, and fish whose potential benefits include accelerating animal growth, enhanced resistance to disease, and better meat quality (Toldra, 2008). 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 technology, when pesticide and other tolerant crops were introduced, it was perceived in different ways both on a regional and an international basis. However, these discussions 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; Snow, 2005). To protect consumer rights along with economical and moral issues with realization of contamination of non-GMOs with GMOs, it has been necessary to prove the safety of GM products in the market. Therefore, several countries, including European Union (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, while legislation in Canada and the United States instead opted for voluntary labeling and requested companies to seek Food and Drug Administration approval before their launch into market (Ahmed, 2002; Guide to U. S. Regulation of Genetically Modified Food and Agricultural Biotechnology Products, 1986). Ninety percent of consumers have less knowledge on what has been quietly introduced into their daily-based food consumption and what impact they might cause in near future. The GMO database (http://www.gmo-compass.org/eng/gmo/db/) shows most of the processed foods available in the grocery Genetically Modified Organisms in Food. http://dx.doi.org/10.1016/B978-0-12-802259-7.00010-5 Copyright © 2016 Elsevier Inc. All rights reserved.

97

98  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

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 GMOs. The need to be able to trace GMOs and their products in food has generated a demand for analytical methods capable of reliably detecting, identifying, and quantifying them. Although there are numerous analytical methods for detection of GMOs, only a limited number of commercialized products may be found (Lin et al., 2001). The review chapter will focus on biosensors as a cutting edge technology based on optical, electrochemical, piezoelectric transducers for GMO detection (screening) and quantification. Table 1 summarizes the detection methods used, target genes and sequences, the limit of detection, and their linear range. (A)

(B)

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.

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

Linearity Range

References

SPR

P35S and T-NOS

1 nM

0.001–2.5 μM

Nica et al. (2004)

SPR

35S from soy, maize

2.5 nM

2.5–25 nM

Mariotti et al. (2002)

SPR

35S from maize and ApoE genes from human blood

2.5 and 50 nM

0–25 nM and 0–250 nM

Feriotto et al. (2002)

SPR a) BIACORE b) SPREETA

GM maize

a) 2.5 nM b) 10 nM

ECL-DNA

CaMV35S

5 nmol/L

5 nmol/L–5 μmol/L

Wang et al. (2004a)

SERS

Genes (cry1A(b) and cry1A(c)) in Bt rice

0.1 pg/mL

1.0 pg/mL−10 ng/mL

Bai et al. (2007)

MS and SERS

35S sequence of Bt-176 maize

11 nM

25–100 nM

Bai et al. (2010)

QCM

pflp gene in tobacco

0.25 ng/mL

0.25 ng/mL–1.0 ng/mL

Kalogianni et al. (2006)

QCM

EPSPS gene in RR soybean

4.7 × 105

DPV

P35S

1 nM/L

0–120 nM/L

Deakln and Bulq (1989)

CaMV35S

4.38 × 10−12 mol/L

1.2 × 10−11–

Mannelli et al. (2003)

ASV

Feriotto et al. (2012)

copies

Guven et al. (2012)

4.8 × 10−8 mol/L LSV

CBH 351 from maize

3 × 102 copies/reaction

0–3 × 105 copies/reaction

Stobiecka et al. (2007)

EIS

CaMV35S

4 pM

0–269 pM

Minunni et al. (2005)

SWV

cryIa/b and MON810 in maize flour

0.6% of transgenes

0.5–0.9%

Karamollaoğlu et al. (2009)

Biosensors for Detection of Genetically Modified Organisms in Food and Feed Chapter | 10  99

STANDARD APPROACHES FOR DETECTION OF GMO Various analytical methods have been developed for reliable determination of presence or absence of GMOs in various food products. Considering EU complex procedures and principles placed for the safety of the GM products, each method supplied by individual must be verified by the Community Reference Laboratory at the Institute of Consumer Health and Protection in Ispra, Italy. Most of these methods undergo validation and confidential analysis of the DNA sequence before appropriate labeling procedures were determined. Although there are hundreds of GMOs developed, the method for detection can be realized by the available information of the modified DNA. The detection method is analyzed based on two important strategies: nucleic base detection on inserted foreign DNA or detection of the novel protein that is specifically expressed in transgenic plants. Protein-based testing methods include one-dimensional sodium dodecyl sulfate gel electrophoresis, western blot, enzyme-linked immunosorbent assays, and lateral flow strip methods that 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. On the other hand, DNA-based testing methods include polymerase chain reaction (PCR), ligase chain reaction, nucleic acid sequence-based amplification, fingerprinting techniques (like restriction fragment length polymorphism, and random amplified polymorphic DNA), and probe hybridization. Because of its high specificity and stability both in qualitative and quantitative analysis, PCR is the most widely preferred method for both raw ingredients and processed food. PCR detection methods use florescent probes during each cycle of amplification process. In order to detect single gene sequence, DNA is usually labeled by fluorophore, organic or inorganic, fluorescent dyes, or quantum dots to enhance the fluorescent yield and to avoid photo bleaching which could hamper the performance of the system. However, these procedures are laborious, expensive, and time-consuming making them not suitable for routine and rapid analyses (Ahmed, 2002; Deisingh and Badrie, 2005; Elenis et al., 2008).

GMO BIOSENSORS Biosensors are analytical devices consisting of a biomolecule as a receptor in close proximity to a transducer, which converts the target analyte–receptor binding event into a measurable signal. Biomolecule can be in the form of an antibody, enzyme, nucleic acid, cell, aptamer, or phage. The measurable signal by the transducer can be based on electrochemical (potentiometric, voltammetric, and conductometric), optical, or piezoelectric methods. Major attributes of biosensor technology include specificity, sensitivity, reliability, portability, real-time analysis, and simplicity of operation (Turner and Newman, 1998). Great progress has been achieved in the development of DNA sensors allowing for a simple, fast, and reliable DNA testing (Sassolas et al., 2008). Considered as the leading edge technology, DNA sensors focuses on simple, rapid, and inexpensive ways of testing and represents an interesting alternative in detection of GMOs (Nica et al., 2004). The raw materials and the processed foods with GMOs contain an additional trait encoded by inserted gene (DNA) which produces additional protein. Therefore, the presence of introduced DNA could be traced by its capture by a specific oligonucleotide probe (recognition layer) that is attached to the surface of the sensor. A schematic of a typical DNA biosensor is shown in Figure 2. Molecular recognition based on hybridization of target DNA sequence with GMO-specific probes has paved a way for the development of various transducers to convert the hybridization event into electrical or optical signals. Based on this, biosensors for GMO detection are categorized into optical, piezoelectric, and electrochemical systems which will be discussed below.

FIGURE 2  Format for detecting DNA sequence in a typical DNA biosensor. DNA probes are immobilized sequences and DNA targets are complementary sequences to be traced. (Reprinted with Permission.)

100  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

Optical Biosensors Optical sensors are potentially the most selective types of sensors involving the interaction between the selective biomolecule binding layer and the transducer. Through optical spectroscopy the detection and quantification of presence of biomolecules is determined. Surface plasmon resonance (SPR) is a powerful optical technique that can detect various biomolecular interactions occurring in the evanescent field generated at the interface of a thin gold-coated prism in contact the analyte solution that flows through. The principle of this instrument lies in determining the refractive index changes occurred at the interface between materials with different refraction indices such as a surface of the prism covered with thin metal layer. In principle, when visible or infrared light is incident on the gold surface at a resonance angle and wavelengths are close to surface plasmon conditions, the intensity of the reflected light is at its minimum. The presence of biomolecules on the surface causes 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 (Campbell and Kim, 2007; Pattnaik, 2005). The first application of an SPR-based biosensor for screening analyses of GMOs was first reported by Mariotti et al. They implemented this sensor in the detection of target sequences P35S and T-NOS that many GMOs have in common. Immobilization of synthesized 25-mer oligonucleotides was carried out first onto 11-mercaptoundecanol and carboxylated dextran-modified gold sensor chip. Hybridization at sensor surface between 25-mer P35S and T-NOS (concentration ranging from 0.001 to 2.5 μM) and their fully complementary oligonucleotides probes were monitored. The detection limit was 1 nM for both P35S and T-NOS with a coefficient of variation (CV) of less than 3%. DNA samples were extracted from real transgenic soy flour with the optimized system. To implement this, DNA samples were first amplified by PCR followed by denaturation with alkaline agents and separation by magnetic particles prior to analysis, and this was the only effective approach to achieve detectable signals for amplified sample in their system (Mariotti et al., 2002). Similar feasibility of SPR-based biospecific interaction analysis for detection of soybean lectin and RR gene sequences was demonstrated by Feriotto et al. Different formats using lectin or Roundup Ready oligonucleotide or PCR-generated probes for detection against target single stranded PCR products were all demonstrated to be useful. Figure 3(A) shows immobilization of Lectin and RR oligonucleotide probes with suitable length (13- and 15-mer) and 21-mer target PCR product detection on SPR chip and (B) representative response (RU) profile upon association, dissociation, and regeneration (Feriotto et al., 2002). Both methods have implemented the use of PCR-generated probes prior to biorecognition event. This was, however, found to have better sensing performance than oligonucleotide probe upon hybridization with its complementary single stranded PCR products in terms of (final RU − initial RU) values, and this method was of far more efficient in detecting and quantifying GMOs (Feriotto et al., 2012). Further research focused on improving the analytical performance of SPR detection in PCR-amplified products. One method for selectively detecting target single strand DNA containing 35S for specific hybridization with complementary P35S probes was developed. Target DNA samples were extracted from soybean powder, maize samples, and pBI121 plasmid. Similar to previously discussed methods, asymmetric PCR amplification was conducted followed by one-step denaturation with alkaline in the presence of formamide at 42 °C. The linear range was up to 25 nM and the detection limit was improved to 2.5 nM with the reproducibility (CV%) of the system less than 5% (Giakoumaki et al., 2003). Wang et al. also proposed a simple and useful denaturation procedure optimized with synthetic oligonucleotides. The method involved a 5-minute denaturation at 95 °C and a 1-minute incubation step with small oligonucleotides tailored to prevent the rehybridizing of the denatured strands. Target 35S promoters from GM maize sample and ApoE genes from human blood were (A)

(a)

(b)

(B)

dsPCR products

ssPCR products

(c)

(d)

Resonance units (RU)

17500

II

III

16500 I 16000

a 1000

b

c 2500 Time (s)

4000

FIGURE 3  (A) Schematic representation of GMO detection using SPR-based biospecific interaction analysis and sensor chips carrying lectin or Roundup Ready oligonucleotides or PCR products. (B) Sensogram response showing increase of resonance units following three (I–III) injections of Roundup Ready PCR products after injecting probe DNA (“a,b” panel) and 50 mM NaOH (“c” panel). (Reprinted with Permission.)

Biosensors for Detection of Genetically Modified Organisms in Food and Feed Chapter | 10  101

used to test the applicability of the method. A linear relationship ranging from 0 to 25 nM and detection limit of 2.5 nM was achieved for the 35S promoter, while a linear relationship up to 250 nM with detection limit of 50 nM was obtained for ApoE genes (Wang et al., 2004a). The group continued to develop a sensitive, reproducible, stable immobilization of DNA probe on commercial sensors BIACORE X™ and SPREETA™ via thiol-derivatized probe/blocking thiol method with detectable concentrations of 2.5 and 10 nM, respectively. Both of the sensors showed high reproducibility (CV% of 1 and 6%). The method was also detectable for DNA samples extracted from GM maize (Wang et al., 2004b). The earliest methods using SPR were based on the real-time monitoring of the hybridization process by change in measurable signal. In addition to its importance, SPR imaging apparatus has become a useful method to observe the hybridization between probes and complementary strand in real time. Spadavecchia et al. studied the hybridization processes of multiple oligonucleotides with complementary oligonucleotides (probes) immobilized on novel photolithographic patterned gold substrates with SPR-based imaging equipment. Thiolated oligonucleotides probes (HS-single stranded (ss)DNA) were self-assembled onto gold traps by a dropping method and the morphology of the surface of substrate were observed with atomic force microscopic technique. The proposed SPR biosensor with suitable generated samples and probe immobilization showed potential in detection of various GMOs with improvements in sensitivity (Spadavecchia et al., 2005). Another solution to make the detection time faster was proposed by Bai et al. As new GMOs are commercialized into the market, there is a demand for efficient and inexpensive assays. Based on this, an optical thin film biosensor chip to detect unique transgenes in GM crops and SNP markers in model plant genomes was reported (Bai et al., 2007). To achieve ultrasensitive detection of SPR biosensors, several approaches have been proposed such as using Au/Ag alloy nanoparticles as sensitized materials. Two Fourier transform–SPR immunosensors, fabricated by antibody/MPA (3-mercaptoproponoic acid)/Au/Ag alloy nanoparticles/HDT and antibody/protein A/MPA/Au/Ag alloy nanoparticles/HDT (1,6-hexanedithiol) sensor chips, respectively, were developed for Cry1Ab protein (insect resistance) detection. The results obtained demonstrated excellent performance for the detection and possessed high specificity and acceptable reproducibility (Figure 4). It was observed that sensor 2 exhibited slightly lower detection limit than sensor 1 due to the protein A immobilized onto sensor 2 (Ming et al., 2015).

FIGURE 4  Top: Schematic diagram of sensor 1 and sensor 2 fabrication and detection procedure. Bottom: FT–SPR spectra of Cry1Ab monoclonal antibody immobilized onto sensor chip 1 (A) and 1000 ng mL_1 Cry1Ab protein detection by sensor 1 (B). Letters indicate reagent added: (a) monoclonal antibody; (d) ethanolamine hydrochloride solution; (f) Cry1Ab protein; (h) NaOH; letters c, e, g, and i were PBS, and b was the endpoint of antibody injection. MPA: 3-mercaptoproponoic acid; HDT: 1,6-Hexanedithiol. (Reprinted with Permission.)

102  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

A similar approach has been used to solve more extended detection of six GM maize lines (Bt11, Bt176, GA21, MON810, NK603, and T25) using optical arrays. Enzyme-based reactions coupled with detection of product by optical means have been developed and are successfully implemented in optical biosensors. In design, the hydrazine-derivatized chip surface of the biosensor was covalently attached with arrays of aldehyde-labeled probes to hybridize with biotinylated PCR amplicons. Later, the system was subjected to a brief incubation with an antibiotin immunoglobulin G horseradish peroxidase conjugate and a precipitable horseradish peroxidase substrate. When biotinylated PCR amplicons hybridize with the probes, a color change on the chip surface (gold to blue/purple) is obtained that can be visualized by unaided eye. This assay is extremely robust, exhibits high sensitivity (numbers) and specificity, and can be extended to detect virtually all GMOs on the chip (Bai et al., 2010). Electroluminescence (ECL) is the emission of light in response to the passage of an electric current or to a strong electric field. The ECL-based optical sensor developed consists of immobilization of biotin probes using streptavidincoated magnetic beads upon platinum working electrodes by activating the magnetic field. The GMO target DNA was hybridized with the immobilized biotin-probe and ruthenium (II) tris–bipyridal (TBR)-probe to form a sandwich complex. Hybridization with TBR reacted with tripropylamine resulted in emission of light for electroluminescence detection. The results indicate the sensor sensitivity of 5 nmol/L of CaMV35S DNA. The calibration curve was stable and linear from 5 nmol/L to 5 μmol/L. The sensor was able to distinctly differentiate between the non-GM tobacco and GM tobacco ECL values obtained which enables this sensor to be a simple, inexpensive, safe, sensitive, and reliable tool (Zhu et al., 2010). Quantitative detection of traces of genetically modified products was demonstrated by chemiluminometric immunosensor array for the detection of recombinant marker proteins expressed in GMOs, that is, 5-enolpyruvylshikimate3-phosphate synthase (EPSPS), neomycin phosphotransferase II (NPTII), and phosphinothricin acetyltransferase (PAT). The monoclonal and polyclonal antibodies raised for specific marker proteins were immobilized on predetermined regions of a glass slide for the sandwich-type immunoassays to be carried out. Photodiodes were located at the bottom of the glass slides in an aligned arrangement to the immobilized antibody sites such that the light signals resulting from the immunoassays could be detected in situ (Figure 5). The sensor array developed was able to detect 1% GMO marked with EPSPS, which was the minimum content over the total content, and 3% GMOs labeled with NPT II or PAT under optimal conditions (Jang et al., 2011). One of the most advanced technology and novel methods developed was surface-enhanced Raman scattering (SERS) spectroscopy. The phenomenon involves the interaction of light, molecules, and metal nanostructures to enhance Raman signals that can resolve the structures down to single molecule level. This is a flexible tool for biological analysis due to its excellent properties for detecting wide varieties of target biomolecules including nucleic acids. The enhancement occurs due to the resonant interaction between optical fields and surface plasmons in the metal. (A) EPSPS 10% 5%

0.06

3%

0.04

2%

0.02

1% 0%

0 0

5

10

15

Detection time (s)

Voltage (V)

Photodiode array placed directly on the bottom of the glass slide

Glass slide with Plastic cover providing the dark immobilized capture compartment consisting of multiple antibodies in a micro- reaction chambers for each analyte spot pattern

(C) PAT

0.08

GMO content:

0.06

5%

0.04

3%

0.02

0,1,2%

0 0

5

10

15

Detection time (s)

20

10%

0.06 0.04

5%

0.02

3% 0,1,2%

5

0

20

10

20

15

Detection time (s)

0.1 (D) Dose-response curves PAT (N=3)

10%

0.1

GMO content:

0.08

0

Voltage (V)

Top plate

(B) NPTII

0.1

GMO content:

0.08

Voltage (V)

Bottom plate

(B) Integrated analytical system

Voltage (V)

0.1

(A) Immunosensor array

0.08

EPSPS (N=4)

0.06

NPTII (N=3)

0.04 0.02 0 0

2

4 6 8 GMO content (%)

10

FIGURE 5  Left: Development of a photodiode-embedded immunosensor array for chemiluminometric detection. The sensor array was placed on the black bottom plate (A) and covered with a top plate to ensure complete darkness within the sensor (B). The top plate also contained a port so reagents could be supplied to the reaction chamber. Right: Dose responses of the chemiluminometric immunosensors to the GMO markers EPSPS, NPT II, and PAT. (Reprinted with Permission.)

Biosensors for Detection of Genetically Modified Organisms in Food and Feed Chapter | 10  103

The principle relies on the incident light excited surface plasmons that radiate a dipolar field, which together leads to redistribution of the electric field around the metal clusters. This effect can cause enhanced excitation intensity from the molecule adsorbed on the metal surface and shift the frequency between incident and excited laser light (Kneipp, 2007). A SERS-barcoded nanosensor was developed on this principle for the detection of Bacillus thuringiensis (Bt) gene-transformed rice expressing insecticidal proteins. The barcoded sensor was designed by fabricating SERS-barcoded nanoparticles with a golden core for optical enhancement, a layer of Raman reporter molecules absorbed onto the surface of gold core for spectroscopic barcoding, and a silica shell for protection and functionalization and conjugation of oligonucleotide strands for targeting DNA strands. Two transgenes (cry1A(b) and cry1A(c)) for Bt rice were used as fusion gene sequence and compared with SPS gene for normal rice to construct a specific SERS-based detection method. After capture, the strands were immobilized on the glass slide and the SERS tags were attached to them for the hybridization reaction to occur. The SERS spectra were finally used to decode the testing results. The detection assay showed good precision, accuracy and sensitivity with a detection limit of 0.1 pg/mL (Chen et al., 2012). A novel method for detection of target 35S DNA was developed by combining magnetic separation and SERS. In the initial step the target-specific oligonucleotide probe immobilized gold-coated magnetic nanospheres were allowed to form oligonucleotide-coated nanoparticles. 5, 5′-dithiobis (2-nitrobenzoic acid) was deposited on the nanorods to form a self-assembled monolayer followed by immobilization of second oligonucleotide probe on the activated nanorod surfaces to form a sandwich layers. Target probes were then allowed to hybridize with the nanoparticles. The system was fully optimized and the SERS analysis was done using 35S sequence of Bt-176 maize sample. The results showed working range of concentration from 25 to 100 nM with a detection limit of 11 nM (Guven et al., 2012). With the advance of new GMOs in the market, new methods of detection were launched. One such example is a design of new biosensor based on de novo and deoxyribozyme computing for GMO detection and classification. Computing is a process that mimics electronic counterparts by utilizing chemical molecular and supramolecular ensembles existing at different states (reversible, switchable) by applying physical/chemical inputs and obtaining readable output. For example, various Boolean logic gates, such as AND, OR, XOR, NOR, NAND, INHIB, XNOR, etc., were realized similarly with biomolecules such as proteins/enzymes, DNA, RNA, and whole cells and were applied for Boolean logic operations (Katz and Privman, 2010). The performances of these gates are based on the presence/absence of the biomolecular entity referred as input signal which triggers the reaction to occur and read the output response. In this study, YesiA E6 deoxyrobozyme gate, referred as one input gate contains E6 deoxyribozyme, ribonucleic acid phosphodiesterase, and stem loop structure attached to the 5′ end. When the system is in the inactive state (noncomplementary sequence), the hydrolysis of dual-labeled substrate is inhibited; however, when the complementary sequence is present, hybridization occurs and the stem loop opens up and permits binding and cleavage of the substrate. This reaction is monitored by fluorophore and quencher labels and increases the fluorescence intensity. This gate exhibited a 14-fold increase in signal intensity due to separation of the fluorophore from the quencher with limit of detection of 22.7 pM in 3 min (May et al., 2008). One of the interesting features of optical biosensors is their sensing can be performed via color reaction with some specific reagents immobilized on the selective layer. Several complexometric reagents and dyes are used for this purpose. Since the era of nanotechnology, nanoparticles have invited vast applications for sensing purposes. Although DNA/ gold nanoparticle-based detection is not new, detection of GMOs using this approach is in its early stages. One such study designed a simple, fast, inexpensive, and disposable dipstick nanoparticle configuration-based DNA for visual detection and confirmation of GMO-related sequences by hybridization. Target sequences (35S and NOS) with probes carrying an oligo (dA) tail were hybridized to biotinylated PCR products and applied on the sample area of the sensor. When dipped in a buffer solution, the buffer migrates and rehydrates the oligo (dT) conjugated gold nanoparticles causing hybridization between poly (dA/dT). At the test zone a red line is formed upon accumulation of nanoparticles, as immobilized streptavidin captures the hybrids (Figure 6). The sensor was tested on soybean powder certified reference material (CRM) with 0.1% GMO content lower the EU threshold (0.9%). The results show that after 35 and 40 amplification cycles for 35S and NOS sequence, GMO was clearly detectable. With real samples from sausages, soy flour, soy beans, and fish meal, red color bands were detectable for 1% GMO content (Kalogianni et al., 2006).

Piezoelectric Biosensors Piezoelectric sensors have also paved way in detection of GMOs based on the increase in mass due to hybridization of probe and the label free target DNA sequence. Quartz crystal microbalance (QCM) is a mass sensitive piezoelectric device based on an oscillatory quartz crystal capable of detecting nanogram changes in mass. The underlying principle is when

104  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

FIGURE 6  (A) Schematic illustration of the principle of the nanoparticle-based DNA biosensor for visual detection of GMO. (B) Dipstick hybridization assay of amplified lectin DNA by using the biosensor. A PCR negative (N), positive samples (S), TZ-test zone and CZ-control zone are shown. (Reprinted with Permission.)

a monolayer or thin film is formed on the quartz wafer sandwiched between two electrodes, an oscillating electric field produces mechanical resonance. The frequency of both electrical and mechanical oscillations is related to interfacial mass changes through the Sauerbrey equation: Δf=



− 2 Δ mnf20 1

A(μρ) 2

where Δf is the oscillation frequency, μ is shear modulus of quartz (2.947 × 1011 g/cm s2), A is the piezoelectrically active crystal area, ρ is the density of the quartz (2.648 g/cm3), and Δm is the mass changes (Bunde et al., 1998; Deakln and Bulq, 1989; Guilbault and Suleiman, 1990). Accordingly, QCM detects real-time monitoring of the hybridization reaction causing increase in the mass and decrease in the resonance frequency. Piezoelectric biosensors work on the principle of detection of GMOs based on the increase in mass due to hybridization of probe and the label free target DNA sequence. Minnuni’s group was the first that had developed an affinity piezoelectric sensor based on DNA hybridization for the detection of GMOs. Streptavidin was covalently linked to thiol/dextran modified gold electrodes on which 5′-biotinylated oligonucleotides probes (25-mer), P35S and T-NOS, were immobilized. The sensor was tested using synthetic complementary oligonucleotides (25-mer) and with a noncomplementary oligonucleotide (23-mer). Real samples of DNA isolated from CRM soybean powder containing 2% of transgenic material and amplified by PCR were also tested. The developed sensor showed advantages in specificity for the synthetic DNA (concentration range from 0.01 to 0.5 μM), rapid screening, and moderate reproducibility (CV of 20%). However, when real DNA samples (pBI121, Gus, 18/20) with diluted concentrations of 40,60, and 100 ng/L were used, the sensor was unable to provide clear quantitative validation between transgenic and nontransgenic samples (Minunni et al., 2001). Another QCM-based DNA sensor has been developed for the detection of 35S promoter and Nos terminator sequences in GMO plants using two immobilization procedures: (a) a thiol/dextran procedure and (b) a thiol-derivatized probe and thiol blocking procedure. Both methods were optimized using synthetic oligonucleotides, samples of plasmidic and genomic DNA isolated from the pBI121 plasmid, CRM, and real samples amplified by PCR. The calibration plot results using complementary P35S showed that thiol dextran procedure had linearity within 0–0.1 μM under working range of 0–0.4 μM. Whereas the thiol-derivatized procedure had linearity from 0 to 0.12 μM under working range of 0–0.25 μM. The percentage CV obtained with PCR amplified DNA from real samples was 11 and 15%, respectively, for sensitivity and linear range close to the synthetic oligonucleotides (values not reported). Both immobilization procedures enabled sensitive and specific detection of GMOs, providing a useful tool for screening analysis in food samples (Figures 7a and b) (Mannelli et al., 2003). Similar work was later aimed at optimizing the coating of the gold quartz crystal surface for probe immobilization by Tombelli et al. However, the sensitivity was found to be higher for the thiolated probes, and the dynamic range was greater with biotinylated probes between 0 and 0.5 μM concentrations of 35S target (Tombelli et al., 2005). Most of the sequences applied for biosensors were amplified using PCR, a method that requires known precise nucleotide sequences flanking both the ends of the target region of DNA. The 35S CaMV promoter and the T-NOS terminator, discussed earlier, are the common transgenic elements used as screening test for GMOs. However, it should be noted that this method is not GMO-specific and any contaminations that lead to false positives should be avoided. Therefore, with existing methods and with the increasing number of GMOs in the market, not all kinds of GMOs can be reliably detected.

Biosensors for Detection of Genetically Modified Organisms in Food and Feed Chapter | 10  105

(A) (a)

(b)

(B)

FIGURE 7  (A) Immobilization procedure. (a) Thiol–dextran immobilization procedure; (b) thiol-derivatized probe/blocking thiol immobilization procedure. (B) Frequency change versus time for the hybridization of 25 bases of complementary DNA oligonucleotide with 25 bases of biotin–ssDNA probe immobilized on a thiol–dextran/streptavidin-coated QCM surface. After the hybridization reaction, the surface has been regenerated dissociating the DNA double helix. (Reprinted with Permission.)

Some transgenic genes that confer certain attributes of the crop in multiple GM varieties or species might also occur across. Several studies were reported in detection of such genes. The Cry1A(b) gene, derived from B. thuringiensis that confers resistance to insects in maize, was detected by a QCM DNA biosensor. Streptavidin-coated gold surface immobilized with probe was employed to detect biotinylated Cry1A(b) gene, MON810 gene from GM maize flour, and a cookie made of similar flour as target sequences. The resonance frequency shift was linearly related to percentage GMO in the range of 0.1–5% of GM MON810 maize flour (Passamano and Pighini, 2006). Another senor was fabricated by immobilization of 21-mer single stranded oligonucleotide (probes) related to the EPSPS gene onto the gold surface of the electrodes via avidin–biotin interaction. RR soybean containing the EPSPS gene confers resistance to the herbicide glyphosate was also detected by QCM biosensor. The hybridization reaction between the probe and the target complementary sequence for the EPSPS gene in DNA samples extracted from animal feed containing 30% RR soybean (amplified and not amplified by PCR) was monitored. The sensor showed the detection limit for genomic DNA in the range of 4.7 × 105 numbers of genome copies contained EPSPS gene (Stobiecka et al., 2007). Another study demonstrated state of the art of QCM sensors for the detection of GMO concentration higher than the 0.9% threshold set by the EU (Minunni et al., 2005). Several other immobilization procedures were reported based on QCM-based DNA biosensor for the detection of CaMV 35S promoter sequence (P35S). First, chemisorption of thiolated probe on gold through thiol–gold interaction and blocking thiol procedure, and second is the covalent attachment of amined probe through gluteraldehyde activation of the 13.56 MHz plasma polymerized ethylene diamine layer. The results showed that thiolated probes provide better immobilization efficiencies and higher sensitivity for the detection of hybridization reaction (Karamollaoğlu et al., 2009). So far most of the reports on GMO biosensors utilize flours, seeds, or processed food as the target raw material. A systematic approach for tracing the transgenes along the whole food processing chain was first reported by Bogani et al. Eight types of processed materials: seeds, crushed seeds, expander, crude flour, proteic flour, crude oil, degummed oil, and lecithin derived from RR soybean was utilized as an initial source during manufacturing process. The gold sensor surface modified with thiol/dextran/streptavidin was immobilized with biotinylated probe and surface complementary target sequences (PCR amplified DNA containing P35S) extracted from the processing chain products were allowed to hybridize and sensor responses were monitored. The findings confirmed a higher signal for all GM-containing samples than for non-GM samples with CV% = 11% (Bogani et al., 2009).

106  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

Electrochemical Biosensors Electrochemical DNA sensors take advantage of interactions between the solid electrode surface, recognition probe, and analyte DNA and offers sensitivity, selectivity, and reliable detection of target sequences upon hybridization. Several approaches to electrochemical detection include direct electron transfer or mediated electron transfer via conductive polymers, specific redox reporters, intercalators, redox dyes, and nanoparticles. Electrochemical techniques that are utilized for GMO detection include cyclic voltammetry, square wave voltammetry (SWV), differential pulse voltammetry (DPV), electrochemical impedance spectroscopy (EIS), linear sweep voltammetry (LSV), and anodic stripping voltammetry (ASV) which will be discussed below (Drummond et al., 2003). Early work on electrochemical hybridization sensor was developed for detecting phosphotransferase neomycine (nptII) gene coding for neomycin/kanamycin resistance trait in transgenic plants by Ligaj et al. The sensor was constructed with pretreated carbon paste electrode and ssDNA-specific probes for the nptII gene were immobilized by applying certain potential and later incubated into a buffer solution containing target DNA (1 μg/mL) and methylene blue indicator. Results show that the square wave voltammetry signal of the indicator decreased in the sample with nptII gene compared with sample devoid of this sequence. At this point, this sensor is only able to distinguish GM and non-GM and limits with sensitivity issues (Ligaj and Oczkowski, 2003). Enzyme-based reactions were employed for detection of GMOs. A study was carried out using hydrolysis of naphthyl phosphate substrate to the electroactive naphthol, and the electrochemical signal was detected by differential pulse voltammetry (Carpini et al., 2004). To implement this, disposable oligonucleotide-modified screen-printed gold electrodes were employed for oligonucleotide probe attachment to the gold surface with the thiol group at the 5′-end. Hybridization experiments were carried by using biotinylated target PCR products and a streptavidin–alkaline phosphatase conjugate was added (Carpini et al., 2004). Another most powerful electrochemical technique “faradic impedance spectroscopy” was used as a stepping stone for extending GMO detection. BCIP/NBT (5-bromo-4-chloro-3-indoyl phosphate/nitro blue tetrazolium) substrate mixture, that acts as a blocking precipitate between the gold surface and a solution containing the [Fe(CN)6]3−/[Fe(CN)6]4− redox pair, was added (Figure 8). The results showed remarkable sensitivity with an estimated detection limit of 1.2 pmol/L (i.e., 7.2 × 106 target molecules in 10 μL of sample solution) for detection of samples from transgenic soy and maize containing 1 and 5% GMO (Lucarelli et al., 2005). Various redox dyes have been used as electron exchange mediators such as ferrocene, ferri/ferrocyanide, ruthenium hexamine, and methylene blue. Methylene blue anchored to the guanine residues of the modified electrode surface was used to differentiate between the signals obtained from the hybrid modified and probe modified SPEs to detect GMOs. SPE electrodes immobilized with oligonucleotide probes were allowed to hybridize with the target PCR sequences (NOS terminator from 2% RR soybean). The SWV technique was used to detect the extent of hybridization between the probe and the target sequence. The results showed detection limits of 1.9 and 2.4 μg/mL for probe and hybrid DNA (Meric et al., 2004). Similar work using gold electrodes was modified with cysteamine selfassembled monolayer which allowed its amino group to covalently attach to the 5′-phosphate end of ssDNA probe with the use of activating reagents water soluble 1-ethyl-3(3′- dimethylaminopropyl)-carbodiimide and N-hydroxysulfosuccinimide. Another study using methylene blue (anchored to the ssDNA probe) was reported to examine the hybridization reactions at the surface of the electrode by voltammetric measurements. The constructed biosensor was tested to detect either 35S promoter or NOS terminator and differentiate between the DNA samples isolated from GM

FIGURE 8  Schematic representation of the impedemetric genosensor (sandwich hybridization assay). The biotinylated hybrid (a,b,c), streptavidin–alkaline phosphatase conjugate coupling (d), and substrate solution (e) product (f) blocked by [Fe(CN)6]3/4− redox probe (g); faradic impedance spectroscopy. BCIP: 5-bromo-4-chloro-3-indoyl phosphate; NBT: Nitro blue tetrazolium. (Reprinted with Permission.)

Biosensors for Detection of Genetically Modified Organisms in Food and Feed Chapter | 10  107

and non-GM soybean. Another interesting feature was the samples were obtained without amplification of detected DNA fragments by PCR (Tichoniuk et al., 2008). Sometimes redox moieties such as [Co (NH3)6]3+ carrying positive charges were reported in construction of electrochemical genosensor. Peptide nucleic acid (PNA) PCR and asymmetric PCR mediated electrostatic interaction of positively charged [Co (NH3)6]3+ complex ions with the negatively charged phosphate backbone of the DNA strands for the detection of target DNA were constructed. Due to the presence of a high percentage of negative charges on DNA–DNA hybrid, the redox moiety [Co (NH3)6]3+ accumulation leads to an increase in the electrical current. This leads to the accumulation of [Co (NH3)6]3+ on the sensor surface depending on the hybrid formed by DNA–DNA, DNA–PNA, and PNA–PNA. The sensor was tested as a combination method for versatile sequences although DNA–PNA also showed approximately similar currents compared with PNA–PNA. Good sensitivity and reproducibility was achieved when tested with soya containing 5% GMO (Kerman et al., 2006). Nanotechnology-based DNA analysis proved to be useful in electrochemical GMO sensors. Mercaptoacetic acidmodified lead sulfide (PbS) nanoparticles were synthesized in aqueous solution and used as specific ssDNA sequence labels for detecting 35S promoter from CaMV. The target DNA sequences were covalently linked on the gold electrode self-assembled with mercaptoacetic acid. Hybridization reaction was monitored by the oxidative dissolution of the PbS nanoparticles in the solution and indirect determination of the lead ion by anodic stripping voltammetry. The detection limit as 4.38 × 10−12 mol/L with a linear range of concentration from 1.2 × 10−11 to 4.8 × 10−8 mol/L (Sun et al., 2008). A similar strategy has been adopted for detection of four modified gene sequences from maize and genetically modified maize by multiplexed labeling with osmium tetroxide bipyridine ([OsO4(bipy)]). This was implemented by mixing the four target strands with the respective oligonucleotides 80% homologous to the central target recognition sequences, and to avoid the latter from binding of [OsO4(bipy)]. Two probes SSIIb and ivrp were used to detect the starch synthase gene IIb and invertase gene of natural maize. Two other probes, CRY that detects the existence of the cry- Ia/b transgene within the sample and the probe 810 that detects the existence of the transgene at the MON810 in the genetically modified maize genome were also used. The aims of the study were to identify (1) the presence of maize (SSIIb and ivrp), (2) the presence of a specific transgene cryIa/b (CRY), and (3) the presence of the specific event Mon 810. SQV was applied to analyze the hybridization event between the target and the probe DNA. This procedure demonstrated no cross reactivity among the target sequences in the same batch medium with good response at higher temperature (50 °C) than 25 and low response to negative controls (Duwensee et al., 2009). While 100% of isogenic and transgenic materials showed SWV signals in less than 10 min, 0.9–0.6% in 30 min and less than 0.5% were not detectable after hybridization (Mix et al., 2012). Several other new genes were detected by Ahmed and coworkers. Maize line CBH 351 (trade name StarLink) contains a modified cry9c gene (confers resistance to feeding damage of lepidopteran insects) from B. thuringiensis subspecies and the bar gene (resistance against the herbicide phosphinotricin) from Streptomyces hygroscopicus. The expression for both the genes is activated by the 35S promoter, and termination of transcription for the cry9c and the bar gene is regulated by the nos adenylation signal and the 35S terminator, respectively. Because the use of StarLink corn was not approved for human consumption, it was considered an urgent need for a novel sensor for detection of these genes. However, it was discovered that traces of cry9c were detected in taco shells and in maize seeds of a non-StarLink variety. The authors for the first time reported an efficient, accurate, and inexpensive rapid detection system which employs loop-mediated isothermal amplification with a higher efficiency than PCR for the detection of maize CBH 351 variety (StarLink). One interesting feature of this method was the anchoring of the amplified samples with a redox active molecule Hoechst 33258 [H33258, 20-(4-hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,50-bi(1H-benzimidazole)] and analyzed by a DNA stick that is integrated with a disposable electrochemical printed chip using LSV. The detection limit of 3 × 102 copies/reaction was achieved for maize CBH 351 (StarLink) (Ahmed et al., 2009). Electrochemical detection of DNA has been one of the new approaches with several advantages especially when enhanced with conducting polymers and carbon nanotubes. Conducting polymers gained great attention in label free detection due to high conductivity, high stability, surface functionality, and electron transfer properties. Due to their high electrical conductivity, good mechanical strength, and stability properties, MWCNTs were immobilized with polypyrrole (PPy), a conducting polymer for CaMV35S DNA hybridization detection. Two techniques were employed, QCM and EIS. The MWCNTs–PPy–DNA system showed “signal on” behavior when the concentration of complementary target DNA was increased, with corresponding decrease in the faradic charge transfer resistance. The sensor with MWCNTs–PPy films obtained limit of detection as low as 4 pM as indicated by QCM data. In principle, this system can be suitable not only for DNA but also for protein biosensor construction (Truong et al., 2010). Regenerative GMO biosensor for detection of CaMV35S promoter gene sequence has been fabricated based on acrylic microspheres and AuNPs composite coated onto SPE with AQMS as a DNA hybridization label using cyclic and differential pulse voltammetry (Figure 9) (Ulianas et al., 2014).

108  SECTION | I  Development, Testing and Safety of Plant and Animal GMO Foods

(A)

(B)

FIGURE 9  (A) (1) The SPE construction. (2) Design mechanism of electrochemical GM DNA biosensor based on acrylic microspheres-modified AuNPs/ SPE. (3) SEM image of acrylic microspheres. (B) Differential pulse voltammograms (A) obtained using various cDNA concentrations of 2.0 × 10−15 (a), 2.0 × 10−14 (b), 2.0 × 10−13 (c), 2.0 × 10−12 (d), 2.0 × 10−11 (e), 2.0 × 10−10 (f), and 2.0 × 10−9 M (g) with 30 min DNA hybridization at 25 °C and GM DNA biosensor linear response range (B). (Reprinted with Permission.)

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