Mary A. Arugula

Enzyme-Based NAND and NOR Logic Gates with Modular Design

The logic gates NAND/NOR were mimicked by enzyme biocatalyzed reactions activated by sucrose, maltose and phosphate. The subunits performing AND/OR Boolean logic operations were designed using maltose phosphorylase and cooperative work of invertase/amyloglucosidase, respectively. Glucose produced as the output signal from the AND/OR subunits was applied as the input signal for the INVERTER gate composed of alcohol dehydrogenase, glucose oxidase, microperoxidase-11, ethanol and NAD(+), which generated the final output in the form of NADH inverting the logic signal from 0 to 1 or from 1 to 0. The final output signal was amplified by a self-promoting biocatalytic system. In order to fulfill the Boolean properties of associativity and commutativity in logic networks, the final NADH output signal was converted to the initial signals of maltose and phosphate, thus allowing assembling of the same standard units in concatenated sequences. The designed modular approach, signal amplification and conversion processes open the way toward complex logic networks composed of standard elements resembling electronic integrated circuitries.

A novel layer-by-layer assembled multi-enzyme/CNT biosensor for discriminative detection between organophosphorus and non-organophosphrus pesticides

Organophosphate compounds are heavily used in agriculture and military activities, while non-organophosphate pesticides are mostly used in agriculture and home defense. Discriminative detection of such toxic compounds is very challenging and requires sophisticated and bulky instrumentation. Meanwhile, multi-enzyme biosensors may offer an effective solution to the problem and may become a versatile analytical tool for discriminative detection of different neurotoxins. In this study, we report for the first time a novel bi-enzyme biosensing system incorporating electrostatically interacted enzyme-armored MWCNT-OPH and MWCNT-AChE along with a set of cushioning bilayers consisting of MWCNT-polyethyleneimine and MWCNT-DNA on glassy carbon electrode for discriminative detection of organophosphorus (OP) and non-organophosphorus (non-OP) pesticides. LbL interfaces were characterized by surface plasmon resonance and electrochemical impedance spectroscopy, demonstrating stepwise assembly and electron conductivity studies. The detection limit was found to be ~0.5 for OP pesticide paraoxon and 1 μM for non-OP pesticide carbaryl, in a wide linear range. The biosensor performance was also validated using apple samples. Remarkable discriminative and straightforward detection between OP and non-OP neurotoxins was successfully achieved with cyclic voltammetry (CV) and UV-vis methods on the MWCNT-(PEI/DNA)2/OPH/AChE biosensor, showing great potential in large screening of OP and non-OP pesticides in practical applications.

Biosensors as 21st Century Technology for Detecting Genetically Modified Organisms in Food and Feed

T 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. 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. 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”), 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. 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. 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). 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. 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.

A biochemical logic approach to biomarker-activated drug release

The present study aims at integrating drug-releasing materials with signal-processing biocomputing systems. Enzymes alanine transaminase (ALT) and aspartate transaminase (AST)—biomarkers for liver injury—were logically processed by a biocatalytic cascade realizing a Boolean AND gate. Citrate produced in the system was used to trigger a drug-mimicking release from alginate microspheres. In order to differentiate low vs. high concentration signals, the microspheres were coated with a protective shell composed of layer-by-layer adsorbed poly(L-lysine) and alginate. The alginate core of the microspheres was prepared from Fe3+-cross-linked alginate loaded with rhodamine 6G dye mimicking a drug. Dye release from the core occurred only when both biomarkers, ALT and AST, appeared at their high pathophysiological concentrations jointly indicative of liver injury. The signal-triggered response was studied at the level of a single microsphere, yielding information on the dye release kinetics.

Novel trends in affinity biosensors: current challenges and perspectives

Molecular biorecognition processes facilitate physical and biochemical interactions between molecules in all crucial metabolic pathways. Perhaps the target analyte and the biorecognition element interactions have the most impactful use in biosensing applications. Traditional analytical sensing systems offer excellent biorecognition elements with the ability to detect and determine the presence of analytes. High affinity antibodies and DNA play an important role in the development of affinity biosensors based on electrochemical, optical and mass sensitive approaches. Advancements in this area routinely employ labels, label free, nanoparticles, multifunctional matrices, carbon nanotubes and other methods to meet the requirements of its own application. However, despite increasing affinity ceilings for conventional biosensors, the field draws back in meeting specifically important demands, such as long-term stability, ultrasensitivity, rapid detection, extreme selectivity, strong biological base, calibration, in vivo measurements, regeneration, satisfactory performance and ease of production. Nevertheless, recent efforts through this line have produced novel high-tech nanosensing systems such as ?aptamers? and ?phages? which exhibit high-throughput sensing. Aptamers and phages are powerful tools that excel over antibodies in sensibility, stability, multi-detection, in vivo measurements and regeneration. Phages are superior in stability, screening for affinity-based target molecules ranging from small to proteins and even cells, and easy production. In this review, we focus mainly on recent developments in affinity-based biosensors such as immunosensors, DNA sensors, emphasizing aptasensors and phage-based biosensors basing on novel electrochemical, optical and mass sensitive detection techniques. We also address enzyme inhibition-based biosensors and the current problems associated with the above sensors and their future perspectives.

Molecular AND logic gate based on bacterial anaerobic respiration.

Enzyme coding genes that integrate information for anaerobic respiration in Shewanella oneidensis MR-1 were used as input for constructing an AND logic gate. The absence of one or both genes inhibited electrochemically-controlled anaerobic respiration, while wild type bacteria were capable of accepting electrons from an electrode for DMSO reduction.

Enzyme-Based Multiplexer and Demultiplexer

A digital 2-to-1 multiplexer and a 1-to-2 demultiplexer were mimicked by biocatalytic reactions involving concerted operation of several enzymes. Using glucose oxidase (GOx) and laccase (Lac) as the data input signals and variable pH as the addressing signal, ferrocyanide oxidation in the output channel was selectively activated by one from two inputs, thus mimicking the multiplexer operation. A demultiplexer based on the enzyme system composed of GOx, glucose dehydrogenase (GDH) and horseradish peroxidase (HRP) allowed selective activation of different output channels (oxidation of ferrocyanide or reduction of NAD(+)) by the glucose input. The selection of the output channel was controlled by the addressing input of NAD(+). The designed systems represent important novel components of future branched enzyme networks processing biochemical signals for biosensing and bioactuating.

Layer-by-layer assembled carbon nanotube-acetylcholinesterase/biopolymer renewable interfaces: SPR and electrochemical characterization.

Developing simple, reliable, and cost-effective methods of renewing an inhibited biocatalyst (e.g., enzymatic interfaces) on biosensors is needed to advance multiuse, reusable sensor applications. We report a method for the renewal of layer-by-layer (LbL) self-assembled inhibition-based enzymatic interfaces in multiwalled carbon nanotube (MWCNT) armored acetylcholinesterase (AChE) biosensors. The self-assembly process of MWCNT dispersed enzymes/biopolymers was investigated using surface plasmon resonance (SPR). The LbL fabrication consisted of alternating cushion layers of positively charged CNT-polyethylenimine (CNT-PEI) and negatively charged CNT-deoxyribonucleic acid (CNT-DNA) and a functional interface consisting of alternating layers of CNT-PEI and negatively charged CNT-acetylcholine esterase (CNT-AChE, pH 7.4). The observed SPR response signal increased while assembling the different layers, indicating the buildup of multiple layers on the Au surface. A partial desorption of the top enzymatic layer in the LbL structure was observed with a desorption strategy employing alkaline treatment. This indicates that the strong interaction of CNT-biopolymer conjugates with the Au surface was a result of both electrostatic interactions between biopolymers and the surface binding energy from CNTs: the closer the layers are to the Au surface, the stronger the interactions. In contrast, a similar LbL assembly of soluble enzyme/polyelectrolytes resulted in stronger desorption on the surface after the alkaline treatment; this led to the investigation of AChE layer removal, permanently inhibited after pesticide exposure on glassy carbon (GC) electrodes, while keeping the cushion layers intact. The desorption strategy permitted the SPR and electrochemical electrode surfaces to be regenerated multiple times by the subsequent self-assembly of fresh PEI/AChE layers. Flow-mode electrochemical amperometric analysis demonstrated good stability toward the determination of acetylcholine with 97.1 ± 2.7% renewability. Our simple, inexpensive approach shows the potential of renewable LbL self-assembled functional interfaces for multiple uses in a wide field of applications such as biosensing, various biotechnological processes, and the food and health industries.

Enzyme catalyzed electricity-driven water softening system

Hardness in water, which is caused by divalent cations such as calcium and magnesium ions, presents a major water quality problem. Because hard water must be softened before use in residential applications, there is great interest in the saltless water softening process because, unlike ion exchange softeners, it does not introduce additional ions into water. In this study, a saltless hardness removal driven by bioelectrochemical energy produced through enzymatic oxidation of glucose was proposed and investigated. Glucose dehydrogenase was coated on a carbon electrode to catalyze glucose oxidation in the presence of NAD⁺ as a cofactor/mediator and methylene green as an electrocatalyst. The results showed that electricity generation stimulated hardness removal compared with non-electricity conditions. The enzymatic water softener worked upon a 6h batch operation per day for eight days, and achieved an average hardness removal of 46% at a high initial concentration of 800 mg/L as CaCO₃. More hardness was removed at a lower initial concentration. For instance, at 200mg/L as CaCO₃ the enzymatic water softener removed 76.4±4.6% of total hardness. The presence of magnesium ions decreased hardness removal because of its larger hydrated radius than calcium ions. The enzymatic water softener removed 70-80% of total hardness from three actual hard water samples. These results demonstrated a proof-of-concept that enzyme catalyzed electricity generation can be used to soften hard water.

Biosensors for Detection of Genetically Modified Organisms in Food and Feed

Genetically modified organisms (GMOs) have gained momentum in improving the agricultural yield through gene transfer systems. Introduction of foreign genes into the host genome for new characteristics demonstrates great progress, however represents a potential risk for the consumers and environment sustainability. Several issues on regulatory approval, safety, and public perception raised concerns that would impact the extent to which GMOs can thrive. Therefore, there is a demand for a simple, sensitive, cost-effective, fast, and reliable detection method capable of operating on the spot. Apart from vast number of available conventional methods, such as enzyme-linked immunosorbent assays or polymerase chain reaction, biosensors are cutting-edge analytical tools that have a great promise and potential in detecting GMOs in wide range of food products, from maize flour to a cookie. In this chapter we discuss the potential application of DNA biosensors for GMO identification/detection based on optical, piezoelectric, and electrochemical transducers reported until now.