Appl Biochem Biotechnol DOI 10.1007/s12010-014-0990-3
Functional Nucleic-Acid-Based Sensors for Environmental Monitoring Arghya Sett & Suradip Das & Utpal Bora
Received: 30 January 2014 / Accepted: 19 May 2014 # Springer Science+Business Media New York 2014
Abstract Efforts to replace conventional chromatographic methods for environmental monitoring with cheaper and easy to use biosensors for precise detection and estimation of hazardous environmental toxicants, water or air borne pathogens as well as various other chemicals and biologics are gaining momentum. Out of the various types of biosensors classified according to their bio-recognition principle, nucleic-acid-based sensors have shown high potential in terms of cost, sensitivity, and specificity. The discovery of catalytic activities of RNA (ribozymes) and DNA (DNAzymes) which could be triggered by divalent metallic ions paved the way for their extensive use in detection of heavy metal contaminants in environment. This was followed with the invention of small oligonucleotide sequences called aptamers which can fold into specific 3D conformation under suitable conditions after binding to target molecules. Due to their high affinity, specificity, reusability, stability, and nonimmunogenicity to vast array of targets like small and macromolecules from organic, inorganic, and biological origin, they can often be exploited as sensors in industrial waste management, pollution control, and environmental toxicology. Further, rational combination of the catalytic activity of DNAzymes and RNAzymes along with the sequence-specific binding ability of aptamers have given rise to the most advanced form of functional nucleicacid-based sensors called aptazymes. Functional nucleic-acid-based sensors (FNASs) can be conjugated with fluorescent molecules, metallic nanoparticles, or quantum dots to aid in rapid detection of a variety of target molecules by target-induced structure switch (TISS) mode. Although intensive research is being carried out for further improvements of FNAs as sensors, challenges remain in integrating such bio-recognition element with advanced transduction platform to enable its use as a networked analytical system for tailor made analysis of environmental monitoring. All authors contributed equally.
A. Sett : S. Das : U. Bora (*) Bioengineering Research Laboratory, Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India e-mail: [email protected] Utpal Bora e-mail: [email protected] S. Das : U. Bora Mugagen Laboratories Pvt. Ltd., Technology Incubation Centre, Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India
Appl Biochem Biotechnol
Keywords Functional nucleic acids . Sensors . Environmental monitoring
Introduction Sensors are analytical tools that react to any physical, chemical, or biological stimuli and generate detectable signals [1]. They are a critical extension of human perception and sensation in many aspects of the life. This is largely because human sensory organs are much less sensitive to the chemical or biological environment than to the physical environment (e.g., light, pressure, temperature, and humidity). Human eye is the most important sensory organ. While focal length of human eye is 3.2 mm and aperture is f2.1–f8.3, it is magnified by various microscopies like compound microscopy and cutting-edge electron microscopy (SEM, TEM, STM). Alike, there is persistent development in sensor technology; new-age sophisticated, sensitive biosensors are based on functional nucleic acid (FNA) dependent platform. There is a plenty of essential chemical or biological compositions which are core, basic, and indispensable elements of the quality of life. The symbiotic relationship between human and its niche leads to consequences into man-made perturbations to environmental systems. So, early detection of emerging environmental pollutants, contaminants, and toxins in natural or artificial environments is important to identify and quantify exposure risks and to select remedial measurements. In an effort to identify and detect these environmental hazards, the ideal sensing technology should be capable to react to and diagnose the type of exposure within seconds. It can cover a wide spectrum of toxic agents, exhibits high sensitivity and reproducibility, requires minimum or no sample preparation, consumes minimal amounts of reagents, and is easy and portable for field operations [2]. The environmental analytical communities always quest for portable, reusable analytical devices which can offer reliable, precise, on-site analysis for a variety of matrices and a host of diverse analytes. Rapid progress in nucleic acid chemistry, in-depth analysis of basic elements of life widened the range of biological recognition candidates and developments in microelectronics. Fiber optics technology optics technology has also expanded the capability of signal-transduction platform. Current trends of chemical detection and quantification strategies like high-resolution gas chromatography, high-resolution mass spectrometryanalysis [3], which are off-line and also often time consuming. Hence, to attain the essential resolution, it does not permit for sufficiently rapid feedback to institute controls or notify mitigation strategies. To encounter the current measurement challenges, cutting-edge sensors are currently designed and developed using novel biological element-based biosensors, autonomous cell-based toxicity monitoring, “lab-on-a-chip” devices rather than conventional/ traditional techniques. Different variability like short lifetime, lack of genetic stability, strong background noise, and strong matrix effect on signal serves as a hindrance to chemical interpretation of traditional biosensors. Hence, in recent era, cell-free system-based biosensors have been emerging to overcome the limitations related to whole cell systems. A few such type of cell-free systems are embodied by single-stranded oligonucleotides composed of DNAzymes and RNAzymes that bind to a variety of molecules with precise specificity [4]. Although first reported in late 1990s, they have been exploited recently as ligands in biosensors with applications in the areas of diagnostics and therapeutics owing to their high selectivity, specificity, high reproducibility, cost-effectiveness, and ease of use. Although nucleic acid oligomers hold less complexity and chemical functionalities compared to proteins, binding affinity toward the target molecule of the functional nucleic acids (FNAs) can also rival that of antibody which is produced in-vivo. There are certain drawbacks of nucleic acids as compared to amino acids which have greater torsional freedom and conformational flexibility. Modification with additional chemical groups or internal structure
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dynamics escalates the conformational freedom and binding capacity of FNAs with their targets [5]. Moreover, diverse chemical groups can be varied to obtain a better and suitable FNA-target contact in complexes of traditionally known nucleoside structure. Merely, a small modification of nucleosides greatly increases the complexity of a library. Altered structure of modified nucleosides offer greater conformational diversity of FNAs and thereby increase the potential of a library. Modified FNA structures can have more stable conformation, which make the FNAs like DNAzyme and RNAzyme more custom-made. Alteration and modifications in the internal structures of DNAzyme and RNAzyme can also increase the affinity for a target. Lastly, modified FNAs can be more resistant to hydrolysis, nuclease resistant, and thermally stable which expands their applicability in the field of sensor technology. The durable, sensitive, and economical FNA-based sensors have now emerged as a novel combat tool against the ever-increasing environmental pollutants such as toxic heavy metals, airborne and waterborne microbes, and other biological toxins in this modern era of industrialization.
Types of FNAs Discovery of ribozymes by Cech and Altman altered the concept that all biocatalysts are protein molecules [6]. Earlier nucleic acids were known as hereditary material for storage of genetic information of life. Functional nucleic acids are not only “blueprint of life” but can also act as catalytic and ligand binding element. FNAs are broadly classified in two categories: (a) catalytic FNAs (DNAzyme and RNAzyme) and (b) non-catalytic FNAs (aptamers and spiegelmers) (Fig. 1).
Fig. 1 Types of various functional nucleic acids (FNAs)
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The catalytic FNAs act in two successive steps: first, recognition of the sequence and then cleavage of the sequence at a specific site. Naturally found RNAzymes can catalyze RNA splicing, cleavage, and peptide bond formation in ribosome machinery [7–9], but artificially derived RNAzymes can catalyze a vast array of reactions such as RNA cleavage, ligation, polymerization, alcohol dehydrogenation, porphyrinmetalation, acyl transfer and degradation of amide, urea, and other small macromolecules [10–17]. Although DNAzymes, lacking 2′ hydroxyl group, are comparatively new, they also can catalyze a variety of reactions ranging from ligation and cleavage reactions of both RNA and DNA, RNA branching and lariat RNA formation, phosphorylation, deglycolation, and other enzyme reaction as of peroxidases. The two most comprehensively well studied RNA-cleaving DNAzymes are 8–17 and 10–23 till date and have the ability to carry out sequence-specific cleavage of all RNA and chimeric RNA/DNA substrates. The complimentary sequence specific against target lies between 8–17 and 10–23 bp catalytic core of deoxyribozyme. The substrate target for each DNAzyme is a chimeric DNA/RNA molecule containing a ribonucleotide (rA/rT/rG/rC) as the cleavage site. These man-made ribozymes, deoxyribozymes, are all key players along with naturally found “molecular wonders” riboswitches. Non-catalytic FNAs like aptamer are also widely exploited in sensor technology. DNA or RNA aptamers can be custom-made, tagged with different fluorophores, quenchers, or metallic nanoparticles for precise and sensitive detection of environmental pollutants. Suitable surface immobilization strategy provides specific adsorption of nucleic acids and also decreases the background noise. Among the physical adsorption and chemical-binding strategies [18], the formation of a gold–thiol bond is comparatively easy and stable and is the most popular for the immobilization of the DNA strand. The carbodiimide bond formed between –COOH and – NH2 is also used to introduce probe DNA. Besides, the biotinylated single-stranded DNA (ssDNA) aptamer can be immobilized on a streptavidin modified electrode through biotinavidin interaction [19]. However, the application of aptamers as sensing molecule is hindered as a result of their susceptibility to nuclease degradation. The incorporation of 2′-modified RNA libraries or modified base, sugar or phosphate-backbone improves the biological half-life significantly. Introduction of chiral principles into the in vitro selection process aids to identify nuclease resistant oligonucleotides. Among few applications, one of the approaches has led to the identification of L-enantiomeric RNA or DNA ligands termed “spiegelmers” that bind to arginine [20], adenosine [21], and vasopressin [22]. Spiegelmers can identify their targets in the same way as aptamers, but they are resistant to enzymatic degradation by nucleases. Noncatalytic FNAs can recognize a wide range of target molecules from biological origin to nonliving molecules. To gain advantage of high sensitivity of catalytic FNAs, aptamers have now been coupled with DNAzymes, RNAzymes, and other catalytic modules to broaden its application as a sensing tool.
In Vitro Selection of FNA from Combinatorial Library Rational methods can be applied to reformulate existing natural ribozymes or to create ribozymes with completely different catalytic and kinetic features [23, 25]. The simplest strategy is composed of splitting a contiguous polynucleotide chain to create a fragmented ribozyme. The fragments can then be reassembled to form an active multicomponent catalytic ribozyme. Simple secondary structure elements can be created by following the Watson-Crick base complementation. These strategies are used routinely to create ribozymes that offer multiple turnover kinetics or altered substrate specificity. However, as with the rational design
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of ribozymes, control over the precise positioning of RNA functional moiety in threedimensional structures remains beyond the reach of current rational design techniques [24, 26]. An alternative or advancement to rational design is the onset of iterative selection methods that isolate catalytic molecules from mutagenized or randomized sequence pools of RNA or DNA. This in vitro selection approach based on the probability that a given pool of random sequence molecules will include individual elements that can perform the function of interest. As an instance, in case of the conserved catalytic core (13 nucleotides) of hammerhead selfcleaving 10–23 ribozyme, it is expected to occur with a frequency of 1 in every 67 million random-sequence RNAs. Therefore, in a pool of 1015molecules, approximately 15 million elements can possess the hammerhead catalytic core, and some of them can efficiently catalyze RNA cleavage. Engineering new catalysts then has been reduced to the synthesis of mutagenized ribozyme pools or random-sequence pools of nucleic acids. But, there is always an uncertainty that a variety of catalytic molecules will be present in a diverse pool of nucleic acids. The more challenging issue is to screen or isolate those rare molecules containing the desired catalytic properties. This problem can be resolved if the elements that perform the desired catalysis can be separated from the remaining pool and subsequently amplified using any of the methods for the replication of RNA and DNA. This process of selective amplification or “in vitro selection” can be used in an iterative fashion to screen rare molecules from large random pools of RNA or DNA. If a significant number of mutations are incorporated to obtain customized candidate during selective amplification, then the process is typically termed as “in vitro evolution” to reflect the similarities between this process and Darwinian evolution. For metal ion-dependent DNAzyme selection, the process is target-dependent. For an instance, the initial DNA pool containing 40–60-nucleotide-long random regions flanked by 2 constant regions is taken. In the middle of this DNA strand, a putative cleavage site is introduced in terms of ribo-adenosine (rA), since a ribonucleotide is ~100,000-fold more susceptible to hydrolytic cleavage than a deoxyribonucleotide. To obtain DNAzymes with high metal ion affinity, the metal concentration is decreased after each round of selection [7, 27] (Fig. 2). To increase the substrate specificity, negative selection steps can be introduced to eliminate sequences that are also active with competing metal ions [28]. After the activity of the DNA pool reached a plateau, the DNAs were cloned and sequenced. After accurate truncation, modification, and rational design of substrate binding sequences, a trans-cleavage DNAzyme is constructed. The strand having rA acts like substrate, and the other strands act like the enzyme. This selection protocol enable nucleic acid enzyme (NAE)-based biosensors to be more tailor-made and more versatile pertaining to various applications [29]. In combinatorial chemistry, any processes including Systemic Evolution of Ligands by EXperimental enrichment (SELEX) involve three steps: synthesis of an aptamer library, selection of screening, and structural analysis of the resulting enzyme-target complex. Aptamers obtained at the end of the SELEX process, high affinity and specificity for a target and their postselection modification can further enhance these properties. In vitro selection initiates with a “random pool” containing typically 1014–15 different DNA sequences (depending on the no. of random nucleotide bases). The pool is designed by flanking a random sequence with two constant regions that are primer binding sites. After the “random pool” is synthesized, it is incubated with the substrate to carry out the desired reaction in the presence of a cofactor. Since in vitro selection normally only utilizes a ssDNA, such ssDNA is then generated from the PCR products, and they are incubated under the desired condition with the substrate to start the next round of selection. After iterative rounds of selection, separation, and amplification, the large initial pool of random sequences can be reduced into a small
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Fig. 2 . Selection, optimization, and reformulation of a Mg2 + -dependent RNA-cleaving DNAzymes. Arrowhead identifies the cleavage site of DNAzyme. The sequences in box are representative of the new hairpin structures that are found in optimized individuals. Encircled nucleotides are conserved (Reprinted with permission Symons [7] Copyright © 1997, American Chemical Society)
population of sequences that are enriched with substrate-specific aptamer. About 10–15 rounds of cyclic selection process are required to select the enriched molecule against the candidate. At the end of the process, the selected population is cloned and sequenced. The resulting sequences are grouped into different families based on sequence similarity, and each family is tested for activity. To identify a spiegelmer, the target of choice has to be prepared in its mirror-image configuration. A standard in vitro selection scheme is performed to isolate an aptamer (Dnucleic acid) that binds to this mirror-image target. After synthesizing the isolated aptamer sequence in its respective L-enantiomeric form, the resulting spiegelmer binds the natural target (in the natural configuration) with comparable affinity [30]. The selection of functional nucleic acids is an execution of the concept of “survival of the fittest molecule,” allowing researchers to find new candidate molecules with desirable activities in the presence of intended targets. Role of “Heavy Metals” in Environmental Pollution The term “heavy metal” is conventionally used to define elements with high density and relative atomic mass above 20 which is toxic to the biological system at low concentrations. Although the term heavy metal has been widely used by chemists and environmental researchers for the last 60 years, such a term has never been defined by International Union of Pure and Applied Chemistry (IUPAC). According to an IUPAC technical report by John Duffus, the term heavy metal is both meaningless and misleading since it has been used indiscriminately to indicate that all metals and semimetals which fall under this category are toxic and hence pollutants. The article further goes on to suggest that the Lewis acid classification on terms of electro-negativity provides a more accurate chemical basis for toxicity assessment of elements without any reference to ‘heaviness’ [31]. Klopman classified elements into two broad classes in terms of electro-negativity where metals with orbital electro-negativities above 1.45 where grouped as Class A while those having electronegativity below −1.88 fell under Class B [32]. The Class B category includes the Lewis acids of large size and high polarizibility like Cu(I), Pd, Ag, Cd, Ir, Pt, Au, Hg, Ti, and Pb(II). Further metals
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with properties intermediary to Class A and B were classified as Borderline metals [33]. Borderline metals included V, Cr, Mn, Fe(II), Co, Ni, Cu(II), Zn, Rh, Pb(IV), and Sn which have been reported to be toxic to biological systems above a threshold concentration. A rational explanation for biological toxicity of certain metals was provided by Nieboer and Richadson. It was hypothesized that Borderline and Class B ions similar in size to Ca (II) can cause structural damage to cell membranes due to their affinity for phosphate groups and nonoxygen carriers present in the membrane [34]. Escalating industrial activities like mining, corrosion, fossil fuel combustion, agriculture, and sludge dumping with increased metallurgical explorations have contributed to the accumulation of Class B and Borderline metals in our environment well and above safety levels. These metals tend to accumulate in the environment silently under our eyes unlike other environmental pollutants like petroleum hydrocarbons or domestic and municipal waste where dumping in the environment is noticeable. Extensive research on environmental pollutants and increased awareness among people has helped to control the levels of such toxic metals. In a concomitant fashion, the increased technological know-how has augmented the development of highly sensitive biosensors for detection and quantification of these toxic metals in our environment at a very nascent stage. Functional nucleic acids like DNAzymes and RNAzymes whose catalytic activity can be regulated by metals has been widely used as sensors in laboratory scale to detect toxic metals in our environmental samples with high specificity and sensitivity. Further, the fact that metals can induce conformational changes in aptamers has been exploited to develop aptasensors for detection of toxic elements even in very low concentrations. FNA-Based Sensors for Detection of Lead (Pb2+) The major source of lead in our atmosphere is exhaust emission from automobiles running on leaded petrol. Such airborne lead contaminates our crop and reaches toxic levels due to biomagnification along the food chain. Some of the other sources of lead in our environment are contaminated water flowing through lead plumbing, lead containing paint and dust, and soil near lead smelters [35]. Elevated levels of lead in the blood (>10 μg/dl in adults and >5 μg/dl in children) can lead to acute neuropathy and renal disorders. Lead interferes with many enzymes like delta-aminolevulinic acid dehydratase, or ALAD, ferrochelataseetc which help in biosynthesis of hemoglobin by binding with the sulphahydryl groups present in these enzymes. Since in many cases lead toxicity remains asymptomatic till almost 50 μg/dl, it is imperative to detect lead in blood and environments samples in trace amount at a nascent stage. Almost a decade ago, a team from University of Illinois, USA, published a series of articles demonstrating the fabrication of DNAzyme based sensors for highly sensitive and specific detection of Pb2+ ions from environmental samples. They showed that catalytic nucleic acids like DNAzymes can be conjugated with gold nanoparticles to form colorimetric biosensor. The nanoparticles were attached to 12mer-DNA molecule having sequence complementary to 17DS region of a 8–17 DNAzyme [36]. Optimization of reaction conditions and improved design of the DNAzyme enabled highly sensitive detection of Pb2+ (around 100 nM) within 10 min at ambient temperature [37, 38]. Later, another group was able to achieve higher sensitivity (1 nM) of lead detection by immobilizing a thiol conjugated catalytic molecular beacon onto a gold surface. Such fluorescent-based colorimetric biosensor could be regenerated to enable multiple cycles of sample testing. This was the first step toward the development of a stand-alone biosensor for in situ detection of Pb [39]. The work was carried forward by Chang et al., who developed a miniaturized lead sensor by integrating a lead specific DNAzyme to a nanocapillary connected microfluidic device to deliver small volume sample into a spatially confined detection
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window [40]. The enhanced sensitivity, portability, and reusability of biosensors having immobilized DNAzymes encouraged further exploration of immobilization platforms. DNAzymes were immobilized on gold nanocapillary membranes [41], PMMA [42], polythiophene [43], and gold electrode surface for fabrication of DNA-Au bio barcodes [44]. The barcode is formed by short oligonucleotides attached to 13-nm gold nanoparticles which hybridize with the substrate strand of DNAzyme. Such electrochemical biosensor can detect very low concentrations of lead (1 nM) in ground and drinking water. In an attempt to develop portable biosensors capable of detecting lead in situ, Mazumdar et al. fabricated a simple dipstick biosensor by immobilizing nanoparticle-DNAzyme conjugate on lateral flow devices for visual detection of lead in paints at a minimum concentration of 5 μM within 10–15 min [45]. Signal amplification by quantitative PCR reaction has enabled development of label-free biosensors for lead. Also, incorporation of quantitative PCR (QPCR) in the system resulted in high sensitivity (with a detection limit of 1 nM) of Pb2+ detection as well as good selectivity for Pb2+ when challenged with coexisting ions [46]. In an alternative approach to develop label-free biosensors, Guo et al. designed K+ stabilised G-quadruplex structures which bound to N-methyl mesoporphyrin IX (NMM) giving high fluorescence signal. In presence of Pb2+, lead could bind competitively to the G-quadruplex to form more compact DNA folds which could not bind to NMM leading to a decrease in fluorescence intensity. This allowed quantitative analysis of Pb2+ by simple “mix and detect” protocol with a detection limit of 1 nM [47]. In a similar method, the dual input requirement of the G-quadruplex was utilized to design a biosensor functioning as an AND gate. The biosensor was composed of a poly-G loop and a GR-5 DNAzyme stem which showed positive signal when Pb2+ and K + exist simultaneously in the system. The combination of hairpin DNA and GR-5 DNAzyme resulted in high selectivity of Pb2+ and a detection limit of 22.8 pM [48]. An electro-chemiluminescent lead biosensor based on GR-5 lead-dependent DNAzyme was reported to detect ultra low concentration of Pb2+ of almost 0.9 pM [49]. In a further innovation, similar G-quadruplex DNAzymes for lead detection were designed and integrated with multiple transducer platforms. Li et al. developed a colorimetric and chemiluminescent lead sensor using a common G-quadruplex DNAzyme named PS2.M. The colorimetric system was found to have a detection limit of 32 nM whereas the chemiluminescent platform gave a better detection limit of 1 nM [50]. Similarly, another group later developed sensitive surface plasmon resonance (SPR) and electrochemical sensing platform for Pb2+ detection using Pb2+ sensitive DNAzyme and a hemin-G Quadruplex nanostructure. The SPR-based system offered ultralow detection limit of 5 fM whereas the electrochemical sensor had a detection limit of 1 pM. All the different sensing platforms provided high selectivity for Pb2+ ions when challenged with coexisting ions [51]. In an attempt to amplify the signal from sensors detecting lead, Zhuang et al. relied on the free energy-driven DNA hybridization chain reaction. They successfully developed a magneto controlled electronic switch to respond to ultra-low concentration of Pb2+ ions in samples (37 pM) utilizing a lead-specific DNAzyme and two ferrocene-labeled DNA hairpins [52]. In a similar approach using streptavidin-conjugated magnetic beads to capture biotinylated GR-5 DNAzyme attached to fluorophore, quantitative analysis of Pb2+ ions was performed by flow cytometric method. Such detection systems do away with conventional cuvette-based measurements which fail to provide accurate data for cloudy environmental samples due to high background as most of the light is absorbed or scattered. A high sensitivity of around 0.6 nM and high selectivity for Pb2+ was reported [53]. DNAzyme-based sensors are almost always associated with an enzyme strand and substrate strand. The use of two different oligonucleotides in one sensor not only made the system complicated to design but also problematic to operate at elevated temperatures. To overcome this, a Chinese group recently designed a DNAzyme-based fluorescent sensor for detection of
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Pb2+ where both the enzyme and substrate strand was combined into a single oligonucleotide chain. The resulting intramolecular duplex structure allows the fluorophore and quencher attached at two ends to remain in close proximity at resting state. In presence of Pb2+, the fluorophore is released leading to increased fluorescence. The sensor was reported to be stable at various temperatures and operate with high selectivity and sensitivity of around 3.1 nM [54]. In a seminal study by Smirnov et al., the group reported for the first time the role of lead ions in sequence-specific folding of DNA. Till then, DNA quadruplex structures were known to be stabilized by divalent cations like K, Na, or NH3+. However, they found that Pb2+ could bind with high affinity and induce folding of thrombin binding aptamers (TBAs) at micromolar concentrations. The study encouraged further research with lead specific aptamers for detection and control of genotoxic effects of lead in the environment [55]. Conjugating a quantum dot and gold nanoparticle to TBA was also reported to be able to detect micromolar levels of lead via optical detection through fluorescent resonant energy transfer [56]. Similar detection systems have been studied for detection of various other heavy metals. Various strategies that evolved with time for detection of Pb2+ from environmental samples are represented as a collage in Fig. 3. Initially, DNAzymes were engineered so that repeated heating and annealing steps for detection are no longer required. Further, the development of fluorescence labeled as well as label-free sensors along with unimolecular (single strand) DNAzyme greatly enhanced the detection speed and simplified on-site detection. Also, the discovery that Pb2+ could bind and induce folding of guanine rich DNA into G-quadruplex structures lead to the development of novel sensors. Using gold nanoparticle conjugated electrodes, DNAzymes anchored to magnetic beads and highly specific aptamers as sensing platform have greatly enhanced the sensitivity by stretching the detection limit to femtomolar concentrations of Pb2 + . FNA Based Sensors for Detection of Mercury (Hg2+) The first report of an epidemic due to exposure to mercury came from Minamata Bay, Japan, in 1956. The poisoning was due to the highly toxic methyl-mercury which came from the nearby
Fig. 3 A schematic representation of the development of various detection platforms for environmental monitoring of Lead. The references are given in superscript while the year denotes the year of publication
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Chisso Corporation factory manufacturing large amounts of acetaldehyde using mercury sulfate as a catalyst. As a side reaction of the process, the toxic methyl mercury was being produced in small quantities and entered the surrounding waters through factory effluents, thus contaminating all fishes in the bay [57]. In the next 4 decades, 2,265 people suffered from crippling neurological diseases out of which 1,784 died. Chisso Corporation extended financial compensation to over 10,000 victims and continued to do so till 2010 [58]. A similar outbreak was reported few years later in Iraq. In the winter of 1971, several people from rural areas of Iraq got admitted with symptoms of mercury poisoning. They were all victims of methyl-mercury poisoning resulting from the extensive use of fungicides (methoxypropylmercury and phenylmercury compounds) in wheat and barley cultivation [59]. The molecular basis of mercury toxicity is based on its ability to irreversibly bind to selenium-containing enzymes or selenozymes like thioredoxin reductase which are essential in controlling oxidative damage in cells [60]. The USFDA and Environmental Protection Agency (EPA) have fixed the maximum allowable level of mercury in food and drinking water to 1 ppm and 1 ppb, respectively [61]. One of the earliest attempts at detecting mercury from environmental samples was made by an American scientist in 1971. He developed a membrane probe-spectral emission type detection system which could detect as low as 0.4 ng of metallic mercury from water samples [62]. Although the apparatus was unique in those days, it has rapidly become out-dated in the modern era of smart, portable biosensors based on advanced materials like functional nucleic acids. Mercury was reported to induce the folding of a DNA strand in the form of a hairpin which enabled its detection by FRET as the fluorophore and the quencher attached at two ends came in close proximity [63]. An array of nucleic acid sensors for mercury were developed utilizing mercury’s strong binding affinity for thymine residues and detect ultralow concentrations of Hg2+. The presence of several thymine residues at the catalytic core of the DNAzyme rendered it inactive. However, even trace amount of Hg2+ was sufficient to form thymine–Hg2+–thymine bonds activating the fluorophore attached DNAzyme beacon leading to increased fluorescence. The system could detect nanomolar concentrations Hg2+ and provide more than 105 times selectivity for Hg2+ over other ions [64]. In a similar study, a detection system composed of polythymine oligonucleotides (T33) and TOTO-3 was designed to detect Hg2+ in pond water and batteries. TOTO-3 is a cyanine dye which exhibits increased fluorescence when bound to double-stranded DNA (dsDNA). The presence of Hg2+ in aqueous solutions was found to induce folding of T33 into a double-stranded structure which could bind to TOTO-3 thereby showing a 1,000-fold increase in fluorescence. The study demonstrated a rapid (
Functional Nucleic-Acid-Based Sensors for Environmental Monitoring Arghya Sett & Suradip Das & Utpal Bora
Received: 30 January 2014 / Accepted: 19 May 2014 # Springer Science+Business Media New York 2014
Abstract Efforts to replace conventional chromatographic methods for environmental monitoring with cheaper and easy to use biosensors for precise detection and estimation of hazardous environmental toxicants, water or air borne pathogens as well as various other chemicals and biologics are gaining momentum. Out of the various types of biosensors classified according to their bio-recognition principle, nucleic-acid-based sensors have shown high potential in terms of cost, sensitivity, and specificity. The discovery of catalytic activities of RNA (ribozymes) and DNA (DNAzymes) which could be triggered by divalent metallic ions paved the way for their extensive use in detection of heavy metal contaminants in environment. This was followed with the invention of small oligonucleotide sequences called aptamers which can fold into specific 3D conformation under suitable conditions after binding to target molecules. Due to their high affinity, specificity, reusability, stability, and nonimmunogenicity to vast array of targets like small and macromolecules from organic, inorganic, and biological origin, they can often be exploited as sensors in industrial waste management, pollution control, and environmental toxicology. Further, rational combination of the catalytic activity of DNAzymes and RNAzymes along with the sequence-specific binding ability of aptamers have given rise to the most advanced form of functional nucleicacid-based sensors called aptazymes. Functional nucleic-acid-based sensors (FNASs) can be conjugated with fluorescent molecules, metallic nanoparticles, or quantum dots to aid in rapid detection of a variety of target molecules by target-induced structure switch (TISS) mode. Although intensive research is being carried out for further improvements of FNAs as sensors, challenges remain in integrating such bio-recognition element with advanced transduction platform to enable its use as a networked analytical system for tailor made analysis of environmental monitoring. All authors contributed equally.
A. Sett : S. Das : U. Bora (*) Bioengineering Research Laboratory, Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India e-mail: [email protected] Utpal Bora e-mail: [email protected] S. Das : U. Bora Mugagen Laboratories Pvt. Ltd., Technology Incubation Centre, Indian Institute of Technology Guwahati, Guwahati, Assam 781039, India
Appl Biochem Biotechnol
Keywords Functional nucleic acids . Sensors . Environmental monitoring
Introduction Sensors are analytical tools that react to any physical, chemical, or biological stimuli and generate detectable signals [1]. They are a critical extension of human perception and sensation in many aspects of the life. This is largely because human sensory organs are much less sensitive to the chemical or biological environment than to the physical environment (e.g., light, pressure, temperature, and humidity). Human eye is the most important sensory organ. While focal length of human eye is 3.2 mm and aperture is f2.1–f8.3, it is magnified by various microscopies like compound microscopy and cutting-edge electron microscopy (SEM, TEM, STM). Alike, there is persistent development in sensor technology; new-age sophisticated, sensitive biosensors are based on functional nucleic acid (FNA) dependent platform. There is a plenty of essential chemical or biological compositions which are core, basic, and indispensable elements of the quality of life. The symbiotic relationship between human and its niche leads to consequences into man-made perturbations to environmental systems. So, early detection of emerging environmental pollutants, contaminants, and toxins in natural or artificial environments is important to identify and quantify exposure risks and to select remedial measurements. In an effort to identify and detect these environmental hazards, the ideal sensing technology should be capable to react to and diagnose the type of exposure within seconds. It can cover a wide spectrum of toxic agents, exhibits high sensitivity and reproducibility, requires minimum or no sample preparation, consumes minimal amounts of reagents, and is easy and portable for field operations [2]. The environmental analytical communities always quest for portable, reusable analytical devices which can offer reliable, precise, on-site analysis for a variety of matrices and a host of diverse analytes. Rapid progress in nucleic acid chemistry, in-depth analysis of basic elements of life widened the range of biological recognition candidates and developments in microelectronics. Fiber optics technology optics technology has also expanded the capability of signal-transduction platform. Current trends of chemical detection and quantification strategies like high-resolution gas chromatography, high-resolution mass spectrometryanalysis [3], which are off-line and also often time consuming. Hence, to attain the essential resolution, it does not permit for sufficiently rapid feedback to institute controls or notify mitigation strategies. To encounter the current measurement challenges, cutting-edge sensors are currently designed and developed using novel biological element-based biosensors, autonomous cell-based toxicity monitoring, “lab-on-a-chip” devices rather than conventional/ traditional techniques. Different variability like short lifetime, lack of genetic stability, strong background noise, and strong matrix effect on signal serves as a hindrance to chemical interpretation of traditional biosensors. Hence, in recent era, cell-free system-based biosensors have been emerging to overcome the limitations related to whole cell systems. A few such type of cell-free systems are embodied by single-stranded oligonucleotides composed of DNAzymes and RNAzymes that bind to a variety of molecules with precise specificity [4]. Although first reported in late 1990s, they have been exploited recently as ligands in biosensors with applications in the areas of diagnostics and therapeutics owing to their high selectivity, specificity, high reproducibility, cost-effectiveness, and ease of use. Although nucleic acid oligomers hold less complexity and chemical functionalities compared to proteins, binding affinity toward the target molecule of the functional nucleic acids (FNAs) can also rival that of antibody which is produced in-vivo. There are certain drawbacks of nucleic acids as compared to amino acids which have greater torsional freedom and conformational flexibility. Modification with additional chemical groups or internal structure
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dynamics escalates the conformational freedom and binding capacity of FNAs with their targets [5]. Moreover, diverse chemical groups can be varied to obtain a better and suitable FNA-target contact in complexes of traditionally known nucleoside structure. Merely, a small modification of nucleosides greatly increases the complexity of a library. Altered structure of modified nucleosides offer greater conformational diversity of FNAs and thereby increase the potential of a library. Modified FNA structures can have more stable conformation, which make the FNAs like DNAzyme and RNAzyme more custom-made. Alteration and modifications in the internal structures of DNAzyme and RNAzyme can also increase the affinity for a target. Lastly, modified FNAs can be more resistant to hydrolysis, nuclease resistant, and thermally stable which expands their applicability in the field of sensor technology. The durable, sensitive, and economical FNA-based sensors have now emerged as a novel combat tool against the ever-increasing environmental pollutants such as toxic heavy metals, airborne and waterborne microbes, and other biological toxins in this modern era of industrialization.
Types of FNAs Discovery of ribozymes by Cech and Altman altered the concept that all biocatalysts are protein molecules [6]. Earlier nucleic acids were known as hereditary material for storage of genetic information of life. Functional nucleic acids are not only “blueprint of life” but can also act as catalytic and ligand binding element. FNAs are broadly classified in two categories: (a) catalytic FNAs (DNAzyme and RNAzyme) and (b) non-catalytic FNAs (aptamers and spiegelmers) (Fig. 1).
Fig. 1 Types of various functional nucleic acids (FNAs)
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The catalytic FNAs act in two successive steps: first, recognition of the sequence and then cleavage of the sequence at a specific site. Naturally found RNAzymes can catalyze RNA splicing, cleavage, and peptide bond formation in ribosome machinery [7–9], but artificially derived RNAzymes can catalyze a vast array of reactions such as RNA cleavage, ligation, polymerization, alcohol dehydrogenation, porphyrinmetalation, acyl transfer and degradation of amide, urea, and other small macromolecules [10–17]. Although DNAzymes, lacking 2′ hydroxyl group, are comparatively new, they also can catalyze a variety of reactions ranging from ligation and cleavage reactions of both RNA and DNA, RNA branching and lariat RNA formation, phosphorylation, deglycolation, and other enzyme reaction as of peroxidases. The two most comprehensively well studied RNA-cleaving DNAzymes are 8–17 and 10–23 till date and have the ability to carry out sequence-specific cleavage of all RNA and chimeric RNA/DNA substrates. The complimentary sequence specific against target lies between 8–17 and 10–23 bp catalytic core of deoxyribozyme. The substrate target for each DNAzyme is a chimeric DNA/RNA molecule containing a ribonucleotide (rA/rT/rG/rC) as the cleavage site. These man-made ribozymes, deoxyribozymes, are all key players along with naturally found “molecular wonders” riboswitches. Non-catalytic FNAs like aptamer are also widely exploited in sensor technology. DNA or RNA aptamers can be custom-made, tagged with different fluorophores, quenchers, or metallic nanoparticles for precise and sensitive detection of environmental pollutants. Suitable surface immobilization strategy provides specific adsorption of nucleic acids and also decreases the background noise. Among the physical adsorption and chemical-binding strategies [18], the formation of a gold–thiol bond is comparatively easy and stable and is the most popular for the immobilization of the DNA strand. The carbodiimide bond formed between –COOH and – NH2 is also used to introduce probe DNA. Besides, the biotinylated single-stranded DNA (ssDNA) aptamer can be immobilized on a streptavidin modified electrode through biotinavidin interaction [19]. However, the application of aptamers as sensing molecule is hindered as a result of their susceptibility to nuclease degradation. The incorporation of 2′-modified RNA libraries or modified base, sugar or phosphate-backbone improves the biological half-life significantly. Introduction of chiral principles into the in vitro selection process aids to identify nuclease resistant oligonucleotides. Among few applications, one of the approaches has led to the identification of L-enantiomeric RNA or DNA ligands termed “spiegelmers” that bind to arginine [20], adenosine [21], and vasopressin [22]. Spiegelmers can identify their targets in the same way as aptamers, but they are resistant to enzymatic degradation by nucleases. Noncatalytic FNAs can recognize a wide range of target molecules from biological origin to nonliving molecules. To gain advantage of high sensitivity of catalytic FNAs, aptamers have now been coupled with DNAzymes, RNAzymes, and other catalytic modules to broaden its application as a sensing tool.
In Vitro Selection of FNA from Combinatorial Library Rational methods can be applied to reformulate existing natural ribozymes or to create ribozymes with completely different catalytic and kinetic features [23, 25]. The simplest strategy is composed of splitting a contiguous polynucleotide chain to create a fragmented ribozyme. The fragments can then be reassembled to form an active multicomponent catalytic ribozyme. Simple secondary structure elements can be created by following the Watson-Crick base complementation. These strategies are used routinely to create ribozymes that offer multiple turnover kinetics or altered substrate specificity. However, as with the rational design
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of ribozymes, control over the precise positioning of RNA functional moiety in threedimensional structures remains beyond the reach of current rational design techniques [24, 26]. An alternative or advancement to rational design is the onset of iterative selection methods that isolate catalytic molecules from mutagenized or randomized sequence pools of RNA or DNA. This in vitro selection approach based on the probability that a given pool of random sequence molecules will include individual elements that can perform the function of interest. As an instance, in case of the conserved catalytic core (13 nucleotides) of hammerhead selfcleaving 10–23 ribozyme, it is expected to occur with a frequency of 1 in every 67 million random-sequence RNAs. Therefore, in a pool of 1015molecules, approximately 15 million elements can possess the hammerhead catalytic core, and some of them can efficiently catalyze RNA cleavage. Engineering new catalysts then has been reduced to the synthesis of mutagenized ribozyme pools or random-sequence pools of nucleic acids. But, there is always an uncertainty that a variety of catalytic molecules will be present in a diverse pool of nucleic acids. The more challenging issue is to screen or isolate those rare molecules containing the desired catalytic properties. This problem can be resolved if the elements that perform the desired catalysis can be separated from the remaining pool and subsequently amplified using any of the methods for the replication of RNA and DNA. This process of selective amplification or “in vitro selection” can be used in an iterative fashion to screen rare molecules from large random pools of RNA or DNA. If a significant number of mutations are incorporated to obtain customized candidate during selective amplification, then the process is typically termed as “in vitro evolution” to reflect the similarities between this process and Darwinian evolution. For metal ion-dependent DNAzyme selection, the process is target-dependent. For an instance, the initial DNA pool containing 40–60-nucleotide-long random regions flanked by 2 constant regions is taken. In the middle of this DNA strand, a putative cleavage site is introduced in terms of ribo-adenosine (rA), since a ribonucleotide is ~100,000-fold more susceptible to hydrolytic cleavage than a deoxyribonucleotide. To obtain DNAzymes with high metal ion affinity, the metal concentration is decreased after each round of selection [7, 27] (Fig. 2). To increase the substrate specificity, negative selection steps can be introduced to eliminate sequences that are also active with competing metal ions [28]. After the activity of the DNA pool reached a plateau, the DNAs were cloned and sequenced. After accurate truncation, modification, and rational design of substrate binding sequences, a trans-cleavage DNAzyme is constructed. The strand having rA acts like substrate, and the other strands act like the enzyme. This selection protocol enable nucleic acid enzyme (NAE)-based biosensors to be more tailor-made and more versatile pertaining to various applications [29]. In combinatorial chemistry, any processes including Systemic Evolution of Ligands by EXperimental enrichment (SELEX) involve three steps: synthesis of an aptamer library, selection of screening, and structural analysis of the resulting enzyme-target complex. Aptamers obtained at the end of the SELEX process, high affinity and specificity for a target and their postselection modification can further enhance these properties. In vitro selection initiates with a “random pool” containing typically 1014–15 different DNA sequences (depending on the no. of random nucleotide bases). The pool is designed by flanking a random sequence with two constant regions that are primer binding sites. After the “random pool” is synthesized, it is incubated with the substrate to carry out the desired reaction in the presence of a cofactor. Since in vitro selection normally only utilizes a ssDNA, such ssDNA is then generated from the PCR products, and they are incubated under the desired condition with the substrate to start the next round of selection. After iterative rounds of selection, separation, and amplification, the large initial pool of random sequences can be reduced into a small
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Fig. 2 . Selection, optimization, and reformulation of a Mg2 + -dependent RNA-cleaving DNAzymes. Arrowhead identifies the cleavage site of DNAzyme. The sequences in box are representative of the new hairpin structures that are found in optimized individuals. Encircled nucleotides are conserved (Reprinted with permission Symons [7] Copyright © 1997, American Chemical Society)
population of sequences that are enriched with substrate-specific aptamer. About 10–15 rounds of cyclic selection process are required to select the enriched molecule against the candidate. At the end of the process, the selected population is cloned and sequenced. The resulting sequences are grouped into different families based on sequence similarity, and each family is tested for activity. To identify a spiegelmer, the target of choice has to be prepared in its mirror-image configuration. A standard in vitro selection scheme is performed to isolate an aptamer (Dnucleic acid) that binds to this mirror-image target. After synthesizing the isolated aptamer sequence in its respective L-enantiomeric form, the resulting spiegelmer binds the natural target (in the natural configuration) with comparable affinity [30]. The selection of functional nucleic acids is an execution of the concept of “survival of the fittest molecule,” allowing researchers to find new candidate molecules with desirable activities in the presence of intended targets. Role of “Heavy Metals” in Environmental Pollution The term “heavy metal” is conventionally used to define elements with high density and relative atomic mass above 20 which is toxic to the biological system at low concentrations. Although the term heavy metal has been widely used by chemists and environmental researchers for the last 60 years, such a term has never been defined by International Union of Pure and Applied Chemistry (IUPAC). According to an IUPAC technical report by John Duffus, the term heavy metal is both meaningless and misleading since it has been used indiscriminately to indicate that all metals and semimetals which fall under this category are toxic and hence pollutants. The article further goes on to suggest that the Lewis acid classification on terms of electro-negativity provides a more accurate chemical basis for toxicity assessment of elements without any reference to ‘heaviness’ [31]. Klopman classified elements into two broad classes in terms of electro-negativity where metals with orbital electro-negativities above 1.45 where grouped as Class A while those having electronegativity below −1.88 fell under Class B [32]. The Class B category includes the Lewis acids of large size and high polarizibility like Cu(I), Pd, Ag, Cd, Ir, Pt, Au, Hg, Ti, and Pb(II). Further metals
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with properties intermediary to Class A and B were classified as Borderline metals [33]. Borderline metals included V, Cr, Mn, Fe(II), Co, Ni, Cu(II), Zn, Rh, Pb(IV), and Sn which have been reported to be toxic to biological systems above a threshold concentration. A rational explanation for biological toxicity of certain metals was provided by Nieboer and Richadson. It was hypothesized that Borderline and Class B ions similar in size to Ca (II) can cause structural damage to cell membranes due to their affinity for phosphate groups and nonoxygen carriers present in the membrane [34]. Escalating industrial activities like mining, corrosion, fossil fuel combustion, agriculture, and sludge dumping with increased metallurgical explorations have contributed to the accumulation of Class B and Borderline metals in our environment well and above safety levels. These metals tend to accumulate in the environment silently under our eyes unlike other environmental pollutants like petroleum hydrocarbons or domestic and municipal waste where dumping in the environment is noticeable. Extensive research on environmental pollutants and increased awareness among people has helped to control the levels of such toxic metals. In a concomitant fashion, the increased technological know-how has augmented the development of highly sensitive biosensors for detection and quantification of these toxic metals in our environment at a very nascent stage. Functional nucleic acids like DNAzymes and RNAzymes whose catalytic activity can be regulated by metals has been widely used as sensors in laboratory scale to detect toxic metals in our environmental samples with high specificity and sensitivity. Further, the fact that metals can induce conformational changes in aptamers has been exploited to develop aptasensors for detection of toxic elements even in very low concentrations. FNA-Based Sensors for Detection of Lead (Pb2+) The major source of lead in our atmosphere is exhaust emission from automobiles running on leaded petrol. Such airborne lead contaminates our crop and reaches toxic levels due to biomagnification along the food chain. Some of the other sources of lead in our environment are contaminated water flowing through lead plumbing, lead containing paint and dust, and soil near lead smelters [35]. Elevated levels of lead in the blood (>10 μg/dl in adults and >5 μg/dl in children) can lead to acute neuropathy and renal disorders. Lead interferes with many enzymes like delta-aminolevulinic acid dehydratase, or ALAD, ferrochelataseetc which help in biosynthesis of hemoglobin by binding with the sulphahydryl groups present in these enzymes. Since in many cases lead toxicity remains asymptomatic till almost 50 μg/dl, it is imperative to detect lead in blood and environments samples in trace amount at a nascent stage. Almost a decade ago, a team from University of Illinois, USA, published a series of articles demonstrating the fabrication of DNAzyme based sensors for highly sensitive and specific detection of Pb2+ ions from environmental samples. They showed that catalytic nucleic acids like DNAzymes can be conjugated with gold nanoparticles to form colorimetric biosensor. The nanoparticles were attached to 12mer-DNA molecule having sequence complementary to 17DS region of a 8–17 DNAzyme [36]. Optimization of reaction conditions and improved design of the DNAzyme enabled highly sensitive detection of Pb2+ (around 100 nM) within 10 min at ambient temperature [37, 38]. Later, another group was able to achieve higher sensitivity (1 nM) of lead detection by immobilizing a thiol conjugated catalytic molecular beacon onto a gold surface. Such fluorescent-based colorimetric biosensor could be regenerated to enable multiple cycles of sample testing. This was the first step toward the development of a stand-alone biosensor for in situ detection of Pb [39]. The work was carried forward by Chang et al., who developed a miniaturized lead sensor by integrating a lead specific DNAzyme to a nanocapillary connected microfluidic device to deliver small volume sample into a spatially confined detection
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window [40]. The enhanced sensitivity, portability, and reusability of biosensors having immobilized DNAzymes encouraged further exploration of immobilization platforms. DNAzymes were immobilized on gold nanocapillary membranes [41], PMMA [42], polythiophene [43], and gold electrode surface for fabrication of DNA-Au bio barcodes [44]. The barcode is formed by short oligonucleotides attached to 13-nm gold nanoparticles which hybridize with the substrate strand of DNAzyme. Such electrochemical biosensor can detect very low concentrations of lead (1 nM) in ground and drinking water. In an attempt to develop portable biosensors capable of detecting lead in situ, Mazumdar et al. fabricated a simple dipstick biosensor by immobilizing nanoparticle-DNAzyme conjugate on lateral flow devices for visual detection of lead in paints at a minimum concentration of 5 μM within 10–15 min [45]. Signal amplification by quantitative PCR reaction has enabled development of label-free biosensors for lead. Also, incorporation of quantitative PCR (QPCR) in the system resulted in high sensitivity (with a detection limit of 1 nM) of Pb2+ detection as well as good selectivity for Pb2+ when challenged with coexisting ions [46]. In an alternative approach to develop label-free biosensors, Guo et al. designed K+ stabilised G-quadruplex structures which bound to N-methyl mesoporphyrin IX (NMM) giving high fluorescence signal. In presence of Pb2+, lead could bind competitively to the G-quadruplex to form more compact DNA folds which could not bind to NMM leading to a decrease in fluorescence intensity. This allowed quantitative analysis of Pb2+ by simple “mix and detect” protocol with a detection limit of 1 nM [47]. In a similar method, the dual input requirement of the G-quadruplex was utilized to design a biosensor functioning as an AND gate. The biosensor was composed of a poly-G loop and a GR-5 DNAzyme stem which showed positive signal when Pb2+ and K + exist simultaneously in the system. The combination of hairpin DNA and GR-5 DNAzyme resulted in high selectivity of Pb2+ and a detection limit of 22.8 pM [48]. An electro-chemiluminescent lead biosensor based on GR-5 lead-dependent DNAzyme was reported to detect ultra low concentration of Pb2+ of almost 0.9 pM [49]. In a further innovation, similar G-quadruplex DNAzymes for lead detection were designed and integrated with multiple transducer platforms. Li et al. developed a colorimetric and chemiluminescent lead sensor using a common G-quadruplex DNAzyme named PS2.M. The colorimetric system was found to have a detection limit of 32 nM whereas the chemiluminescent platform gave a better detection limit of 1 nM [50]. Similarly, another group later developed sensitive surface plasmon resonance (SPR) and electrochemical sensing platform for Pb2+ detection using Pb2+ sensitive DNAzyme and a hemin-G Quadruplex nanostructure. The SPR-based system offered ultralow detection limit of 5 fM whereas the electrochemical sensor had a detection limit of 1 pM. All the different sensing platforms provided high selectivity for Pb2+ ions when challenged with coexisting ions [51]. In an attempt to amplify the signal from sensors detecting lead, Zhuang et al. relied on the free energy-driven DNA hybridization chain reaction. They successfully developed a magneto controlled electronic switch to respond to ultra-low concentration of Pb2+ ions in samples (37 pM) utilizing a lead-specific DNAzyme and two ferrocene-labeled DNA hairpins [52]. In a similar approach using streptavidin-conjugated magnetic beads to capture biotinylated GR-5 DNAzyme attached to fluorophore, quantitative analysis of Pb2+ ions was performed by flow cytometric method. Such detection systems do away with conventional cuvette-based measurements which fail to provide accurate data for cloudy environmental samples due to high background as most of the light is absorbed or scattered. A high sensitivity of around 0.6 nM and high selectivity for Pb2+ was reported [53]. DNAzyme-based sensors are almost always associated with an enzyme strand and substrate strand. The use of two different oligonucleotides in one sensor not only made the system complicated to design but also problematic to operate at elevated temperatures. To overcome this, a Chinese group recently designed a DNAzyme-based fluorescent sensor for detection of
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Pb2+ where both the enzyme and substrate strand was combined into a single oligonucleotide chain. The resulting intramolecular duplex structure allows the fluorophore and quencher attached at two ends to remain in close proximity at resting state. In presence of Pb2+, the fluorophore is released leading to increased fluorescence. The sensor was reported to be stable at various temperatures and operate with high selectivity and sensitivity of around 3.1 nM [54]. In a seminal study by Smirnov et al., the group reported for the first time the role of lead ions in sequence-specific folding of DNA. Till then, DNA quadruplex structures were known to be stabilized by divalent cations like K, Na, or NH3+. However, they found that Pb2+ could bind with high affinity and induce folding of thrombin binding aptamers (TBAs) at micromolar concentrations. The study encouraged further research with lead specific aptamers for detection and control of genotoxic effects of lead in the environment [55]. Conjugating a quantum dot and gold nanoparticle to TBA was also reported to be able to detect micromolar levels of lead via optical detection through fluorescent resonant energy transfer [56]. Similar detection systems have been studied for detection of various other heavy metals. Various strategies that evolved with time for detection of Pb2+ from environmental samples are represented as a collage in Fig. 3. Initially, DNAzymes were engineered so that repeated heating and annealing steps for detection are no longer required. Further, the development of fluorescence labeled as well as label-free sensors along with unimolecular (single strand) DNAzyme greatly enhanced the detection speed and simplified on-site detection. Also, the discovery that Pb2+ could bind and induce folding of guanine rich DNA into G-quadruplex structures lead to the development of novel sensors. Using gold nanoparticle conjugated electrodes, DNAzymes anchored to magnetic beads and highly specific aptamers as sensing platform have greatly enhanced the sensitivity by stretching the detection limit to femtomolar concentrations of Pb2 + . FNA Based Sensors for Detection of Mercury (Hg2+) The first report of an epidemic due to exposure to mercury came from Minamata Bay, Japan, in 1956. The poisoning was due to the highly toxic methyl-mercury which came from the nearby
Fig. 3 A schematic representation of the development of various detection platforms for environmental monitoring of Lead. The references are given in superscript while the year denotes the year of publication
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Chisso Corporation factory manufacturing large amounts of acetaldehyde using mercury sulfate as a catalyst. As a side reaction of the process, the toxic methyl mercury was being produced in small quantities and entered the surrounding waters through factory effluents, thus contaminating all fishes in the bay [57]. In the next 4 decades, 2,265 people suffered from crippling neurological diseases out of which 1,784 died. Chisso Corporation extended financial compensation to over 10,000 victims and continued to do so till 2010 [58]. A similar outbreak was reported few years later in Iraq. In the winter of 1971, several people from rural areas of Iraq got admitted with symptoms of mercury poisoning. They were all victims of methyl-mercury poisoning resulting from the extensive use of fungicides (methoxypropylmercury and phenylmercury compounds) in wheat and barley cultivation [59]. The molecular basis of mercury toxicity is based on its ability to irreversibly bind to selenium-containing enzymes or selenozymes like thioredoxin reductase which are essential in controlling oxidative damage in cells [60]. The USFDA and Environmental Protection Agency (EPA) have fixed the maximum allowable level of mercury in food and drinking water to 1 ppm and 1 ppb, respectively [61]. One of the earliest attempts at detecting mercury from environmental samples was made by an American scientist in 1971. He developed a membrane probe-spectral emission type detection system which could detect as low as 0.4 ng of metallic mercury from water samples [62]. Although the apparatus was unique in those days, it has rapidly become out-dated in the modern era of smart, portable biosensors based on advanced materials like functional nucleic acids. Mercury was reported to induce the folding of a DNA strand in the form of a hairpin which enabled its detection by FRET as the fluorophore and the quencher attached at two ends came in close proximity [63]. An array of nucleic acid sensors for mercury were developed utilizing mercury’s strong binding affinity for thymine residues and detect ultralow concentrations of Hg2+. The presence of several thymine residues at the catalytic core of the DNAzyme rendered it inactive. However, even trace amount of Hg2+ was sufficient to form thymine–Hg2+–thymine bonds activating the fluorophore attached DNAzyme beacon leading to increased fluorescence. The system could detect nanomolar concentrations Hg2+ and provide more than 105 times selectivity for Hg2+ over other ions [64]. In a similar study, a detection system composed of polythymine oligonucleotides (T33) and TOTO-3 was designed to detect Hg2+ in pond water and batteries. TOTO-3 is a cyanine dye which exhibits increased fluorescence when bound to double-stranded DNA (dsDNA). The presence of Hg2+ in aqueous solutions was found to induce folding of T33 into a double-stranded structure which could bind to TOTO-3 thereby showing a 1,000-fold increase in fluorescence. The study demonstrated a rapid (
