Trends in Green Analytical Chemistry for Aquatic Environment Pollution Analysis

Trends in Analytical Chemistry, Vol. 29, No.
11, 2010
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Green analytical chemistry in the determination of organic pollutants in the aquatic environment
, Sandra Pe rez, Carlos Gonc Marinella Farre alves, M.F. Alpendurada, ` Barcelo Damia
We present the latest advances in green analytical chemistry for application to organic-pollution analysis in aquatic environments. We review the main strategies to reduce toxic reagents, solvent wastes and energy consumption. We pay special attention to new approaches to environmental analysis, allowing automation, miniaturization, and on-site, on-line and direct analysis (e.g., biosensors). 2010 Elsevier Ltd. All rights reserved.
Keywords: Aquatic environment; Automation; Direct analysis; Environmentally-friendly method; Green analytical chemistry; Miniaturization; Biosensor; Organic pollution; Toxic reagent; Solvent waste
1. Introduction
*, Marinella Farre rez, Sandra Pe ` Barcelo Damia Department of Environmental Chemistry, IDAEA-CSIC, c/Jordi Girona, 18-26, 08034 Barcelona, Spain Carlos Gonc alves, M.F. Alpendurada gua da IAREN, Instituto da A Regia o Norte Rua Dr. Eduardo Torres, 229 4450-113 Matosinhos, Portugal, and Laboratory of Hydrology, Faculty of Pharmacy, University of Porto/Rua An bal Cunha, 164/4050-047 Porto, Portugal ` Barcelo Damia Catalan Institute of Water Research, ICRA, c/Emili Grahit, 101, Edifici H2O, Parc Cient fic ` gic de la Universitat i Tecnolo de Girona, E-17003 Girona, Spain
Corresponding author. Tel.: +34 934006100x435; Fax: +34 932045904. E-mail: mfuqam@iiqab.csic.es, mfuqam@cid.csic.es
Due to scientic and public concern about environment pollution, environmentalfriendly practices have been introduced in different areas of society, especially research. The main objectives of green analytical chemistry (GAC) are to obtain new analytical technologies or to modify an old method to incorporate procedures that use less hazardous chemicals or use smaller amounts of hazardous chemicals. Malissa rst presented the basis of GAC in Paris, and, 15 years ago, de la Guardia et al. [1] rst introduced the topic of environmental analytical chemistry as a model of analytical practices in an integrated approach to analytical chemistry that also considered environmental side effects of analytical practices. Since then, this concept has been gaining interest [2], but it was in recent years that a great effort of development was made to obtain analytical technologies able to do direct analysis, using miniaturized equipments, reduced amounts of solvents, and on site, and reducing energy costs and wastes. These improvements were linked to advances in other research areas (e.g., microelectronics, material sciences, biochemistry, and, recently, nanotechnology).
This review provides the state of the art of GAC for environmental analysis of organic pollution in aquatic environments with special emphasis on strategies for reducing, or even eliminating, sample pretreatment with novel approaches based on biosensors.
2. Sample preparation Sample pre-treatment can be considered the most polluting step of the whole analytical process because it deals with the crude sample, in which the analytes may exist in 1000-fold smaller quantities than bulk constituents, so, often, the use of organic solvents is required to enrich the target compounds selectively and remove potentially interfering matter. An ideal GAC protocol avoids sample pre-treatment. However, in the majority of the cases, this is not feasible, so a plethora of strategies have been developed to obtain greener approaches. 2.1. Techniques for reducing solvent use Sample-preparation techniques for environmental analysis can be classied according to the types of matrix to be analyzed, so these strategies can be grouped into those for solid samples and 1347
0165-9936/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.trac.2010.07.016
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Trends in Analytical Chemistry, Vol. 29, No. 11, 2010
liquid samples. In this context, the main techniques are supercritical uid extraction (SFE), sub-critical water extraction (SWE) and pressurized liquid extraction (PLE), followed by solid-phase microextraction (SPME), stir-bar sorptive extraction (SBSE), microextraction in packed syringe (MEPS), liquid-phase microextraction (LPME), single-drop microextraction (SDME) and dispersive liquid-liquid microextraction (DLLME). We review these techniques in the following sub-sections. 2.1.1. Solid-phase microextraction. In recent years, several authors have focused on improving SPME sensitivity by approaching exhaustive extraction of the analytes by creating a temperature gap between sample and coating. This objective was successfully attained using an internally-cooled polydimethylsiloxane (PDMS)-coated ber with CO2 or chilled alcohol for cooling the ber. Polycyclic aromatic hydrocarbons (PAHs) and; polychlorodibenzo-p-dioxins and polychlorodibenzo-furans (PCDD/Fs) were analyzed, respectively, in contaminated soils [3]. The efciency of this arrangement is optimal, since the sample can be heated to favor the release of the analytes and the ber is cooled to increase their partition coefcients onto the adsorbent. Increasing the polarity of the polymeric coating to be more appropriate for polar analytes is another research eld in SPME. Sol-gel coatings have been developed to overcome the limitations of commercial phases with respect to solvent instability and swelling, low operating temperature, stripping off the coating and inadequate polarity [4]. Several coating compositions have been proposed: amino-functionalized SPME ber using 3-(trimethoxysilylpropyl) amine as sol-gel precursor [5]; sol-gel titania poly(tetrahydrofuran), which proved very suitable for a wide range of analytes and resistant to extreme pH conditions [6]; (3-aminopropyl) triethoxysilane- and diethoxydiphenylsilane-based sol-gel with excellent thermal (400C) and chemical stability [7]; and, porous polypirrole for the extraction of polar, aromatic and anionic compounds (e.g., anatoxin-a, ochratoxin-a, phenols and even nitrite, nitrate and chloride in water samples) [8]. Other approaches use amphiphilic and hydrophilic oligomers, calyx arenes, several crown ethers [9] and nanomaterials {e.g., oxidized multi-walled carbon nanotubes (MWCNTs) [10] and single-walled CNTs (SWCNTs) [11]}. The improved selectivity that is achieved with molecularly imprinted polymers (MIPs) as SPME coatings greatly simplies the analysis of complex environmental and biological matrices, saving many steps of fractionation and clean up of the extracts and maintenance of the equipments, thus reducing labor and solvent consumption [8,12].
2.1.2. Stir-bar sorptive extraction. Using SBSE, the extraction process takes place in a magnetic stir bar 12 cm long coated with a layer of adsorbent polymer. Until now, PDMS is the only adsorbent commercially available with 0.31.0 mm thickness (25125 lL volume) [13]. Although the basic principles of SPME and SBSE are identical and the extraction phase is generally the same, the amount of PDMS is 50250 times larger in SBSE. This feature allowed the preconcentration efciency to be improved compared to SPME, which is its main advantage [13]. Nevertheless the technique did not gain wider acceptance in the eld of environmental analysis, as it was limited by the type of extraction coating that is unsuitable for direct preconcentration of polar pollutants [14]. The most popular applications are therefore analysis of persistent organic pollutants (POPs) [e.g., PAHs, pesticides, polychlorinated biphenyls (PCBs) and polybrominated diphenyl ethers (PBDEs)], even though some successful applications in the eld of emerging environmental contaminants have emerged {e.g., determination of alkylphenols [15,16], sunscreen agents [17], endocrine disruptors [18] and some pharmaceuticals [19]}. The recent research focused mainly on the development of novel extraction phases and SBSE instrumentation [13]. Advances in terms of SBSE conguration include the SBSE arrangement called dual-phase twisters comprising a hollow PDMS tube sealed at both ends by magnetic stoppers [20]. The inner compartment can be lled with different adsorbents (e.g., activated carbons), which confer the capacity to enrich not only apolar compounds but also rather polar ones, through adsorption and absorption mechanisms. Roy et al. created an on-site extraction system for the analysis of 24 PAHs comprising an SBSE stirrer and eld apparatus (EM 640 S from Bruker) [21]. Among the main advantages of this device were sensitivity at the sub-ppt level, unattended operation and minimized risk of cross-contamination and loss of analytes.
2.1.3. Microextraction in packed syringe. MEPS is a miniaturized solid-phase extraction (SPE) technique introduced in 2004 by the group of Abdel-Rehim [22,24]. Approximately 1 mg of the solid packing material is immobilized inside a syringe barrel (100250 lL) as a plug or tiny cartridge. For extraction, 10250 lL of an aqueous sample is withdrawn by the syringe into an autosampler [24]. It can be connected on-line to gas chromatography (GC) or liquid chromatography (LC) without any modication. The adsorbent can be used for repeated extractions (up to 400) before the syringe is discarded [24]. Furthermore, MEPS reduces the handling time by about 30 and 100 times compared to SPME and SBSE, respectively, saving time and resources. The analytes are eluted with a very small
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amount of solvent (2050 lL) directly into the instrument injector. In addition, all types of adsorbents can be used in MEPS {i.e. reversed phase, normal phase, ion exchange, MIPs and random access material (RAM) [23,25]}. While, for in-tube SPME, a capillary or needle is internally coated with a polymeric lm, Pawliszyn called a similar assembly a needle-trap device (NTD), differing in the lumen being packed with an extraction phase [26]. Generally, the needle-shaped extraction devices are miniaturized systems, in which a section of the needle is coated with a polymer on the inside, and packed with particulate materials or lled with a bundle of coated microbers [27]. The needle-shaped extraction devices are especially suitable for the enrichment of volatile compounds, since a continuous ow of the gaseous sample can be supplied through the extraction needle, but they can be also employed for water samples [27]. NTDs, and generally all needle-shaped extraction devices, are more robust than SPME and their sorbent capacity is bigger, making it possible to perform exhaustive extractions. Qi et al. investigated the feasibility of a ber-packed extraction device for the determination of aromatic pollutants in water samples. Three nanobers of poly(styrene-co-methacrylic acid), poly(styrene-costyrenesulfonate) and polystyrene were employed as the extraction medium and packed into a section of a pipette tip [28]. Nanobers of Zylon and Technora were chosen by some authors because of their excellent thermal stability and resistance to typical organic solvents [29]. HR-1 (100%-methyl-polysiloxane) and HR-17 (50%phenyl-50%-methyl-polysiloxane) were employed as the polymer coatings [27]. Saito et al. published a very good review on the principles, construction and special applications of berpacked needle-type extraction devices [27]. Another innovation involves packing a common adsorbent or a monolithic methacrylate polymer, as reported by Blomberg, in a conventional micropipette tip or 96-well-plate system [23]. The latter allows great improvement in analytical throughput. A relevant green feature displayed by MEPS, berpacked needle devices and other techniques discussed above, is their reusability, which translates into reduced analytical costs and minimized waste. 2.1.4. Thin-lm microextraction. Bruheim et al. proposed an extraction tool called thin-lm microextraction (TFME), comprising a PDMS lm that is cut into the shape of a house and secured in its centre by a stainless steel wire, allowing it to rotate [30]. Due to its bigger surface area, it possesses higher extraction capacity than the SPME ber and equilibration times are faster. Nevertheless, it remains quite a manual procedure and the desorption step is not so well established. TFME was used
both for grab sampling and on-site passive sampling of PAHs in Hamilton Harbor [4]. 2.1.5. Liquid-phase extraction. Liquid-liquid microextractions (LLMEs) include a great variety of congurations and solvent types that have been deployed to counteract each others limitations and enlarge the analytical scope. Below, we discuss the SDME, organic solvent lm (OSF), continuous-ow microextraction (CFME) and hollow-ber liquid-phase microextraction (HFLPME) techniques, while we deal with DLLME in a separate sub-section, given the enormous expectations that is has created. SDME is a conceptually very interesting extraction and enrichment technique, rst introduced in 1996. It is nearly solventless, since it typically requires just 13 lL of an organic solvent immiscible with water. The solvent drop is suspended from the tip of a micro syringe and held in contact with the sample or its headspace until an equilibrium partition of the analytes is achieved [8]. Its attractiveness rests on the following features: it is inexpensive, does not require dedicate equipment, is easy to operate and provides an extract that can be directly analyzed by GC or LC. Unfortunately, it remains quite a manual technique, although automation seems feasible. Mechanical instability of the drop and lack of appropriate solvent characteristics for certain analytes are the main limitations. On these grounds, current research focuses on extraction with new solvents for enrichment of polar analytes and active extraction by chemical modication of the extracting drop [8]. New, greener solvents [e.g., ionic liquids (ILs)] are being tested and employed for extraction of polar analytes, and water is used in headspace extraction. Adding chemical reactivity to the extractive drop by doping it with a derivatization or complexation reagent opens up many possible applications. Interesting strategies are used in SDME {e.g., noble metal-containing aqueous drops for enrichment of hydride-forming elements, and acidic and alkaline aqueous drops for extraction of ionizable inorganics (e.g., ammonia and cyanide) [31]}. To circumvent the mechanical instability and limited extracting surface area of SDME, Shen and Lee developed the OSF approach to that we might call inside-needle solvent lm extraction [32]. It relies on a continuous renewal of the solvent lm generated inside the needle by movements of the plunger. Also, to tackle solvent-drop instability, HFLPME was developed. In the two-phase model, the organic solvent is immobilized inside a porous polypropylene capillary tube and placed in contact with the water sample. The analytes tend to partition into the solvent through the pores of the capillary. In the three-phase model, the analytes cross the organic solvent embedded in the pores of the semi-permeable membrane and concentrate in the third phase inside the lumen by modifying their solubility.
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HFLPME makes a notable contribution to green chemistry, since it is environment friendly, consumes little solvent, provides clean-up features due to its selectivity, has high enrichment factors and recoveries, relies on low-cost materials, and is versatile for different sorts of analytes and matrices [33]. 2.1.6. Dispersive liquid-liquid microextraction. DLLME, rst introduced by Berijani and co-workers in 2006, involves three phases: the sample; the extraction solvent with high density and immiscible with the sample; and, a disperser solvent miscible with both. A mixture of a few lL of extraction solvent and $1 mL of disperser solvent, in pre-determined proportions, is injected into the sample. Instantaneously, a cloudy solution made of thousands of very tiny drops is formed. The intimate contact of the organic extraction solvent with the sample favors the transfer of the analytes by simple solvent partition [34]. The extraction solvent is separated by centrifugation and then analyzed. The main advantages attributed to DLLME are simplicity of operation, rapidity, low cost, high recovery, high enrichment factor, and being environmentally benign [35,36]. Nerin et al. [8] compiled a comprehensive collection of DLLME applications where water of various environmental origins was the predominant matrix. DLLME has been applied successfully to the analysis of antimicrobials [35], organophosphorous, organotin and pyrethroid pesticides, triazine and amide herbicides, fungicides, PAHs, organophosphorous ame retardants, PCBs, PBDEs, trichloromethane, chlorobenzenes, phthalate esters, pharmaceuticals and heavy metals in environmental samples coupled to GC, LC and atomic absorption spectroscopy [34]. DLLME applications have extended to the analysis of emerging pollutants (e.g., fragrances and antidepressant drugs) [37]. Such widespread acceptance is a good indicator of its analytical usefulness. DLLME has already gained a great deal of prestige, but its range of applications is expected to broaden in the future in three directions, all having green features: (1) application to matrices more complex than environmental water; (2) wider selection of extraction solvents other than halogenated hydrocarbons; and, (3) coupling with sophisticated, highly sensitive detection instruments [37]. 2.1.7. Supercritical uid extraction. SFE relies on the use of a gas compressed at a pressure and temperature above the critical point (Pc). It comprises a dense gas state in which the uid combines hybrid properties of liquid and gas. Supercritical CO2 has very useful properties: high diffusivity to extract organic compounds from solid matrices; not ammable; moderate and easily accessible critical point conditions; low cost; decompression directly to the atmosphere; and, harmless to the environment. 1350
However, there are at present limitations in the extraction of polar compounds. Addition of an organic modier or selecting a different supercritical uid is possible, but some ideal properties of CO2 are lost. The appearance of a technique, PLE, claiming the advantages of both SFE and liquid-solid extraction with high extraction efciency, diverted attention from SFE. However, the short development period of SFE leaves some hope that new improvements might appear in this eld. Recently, SFE was applied to monitor priority and emerging pollutants {e.g., inorganic and organolead compounds in sand, urban dust and sediments [38], pesticides in soil [39], polyhalogenated pollutants (PCBs, PBBs, PBDEs, (PCDD)/Fs and OCPs) [40]}. 2.1.8. Subcritical water extraction. Also known as pressurized hot water extraction (PHWE), SWE uses an environment-friendly solvent. The possibility of adjusting the dielectric constant of the supercritical water, and thus the solubility of organic substances, gives the opportunity to extract polar and mid-polar compounds. Nevertheless, it is unsuitable for extracting very hydrophobic, thermolabile and hydrolizable substances [8]. In the environmental eld, SWE has been widely used in the extraction of soil samples. Like SFE, SWE has found legitimate application in bioavailability and bioaccessibility studies in support of risk assessment of contaminated land. Latawiec and Reid evaluated two emerging non-exhaustive extraction techniques (SWE and Brij 700 extraction) to reect PAH bioaccessibility to microorganisms, compared with methodologies demonstrated before. Whilst the use of cyclodextrins was the best predictor of the bioaccessible fraction for the majority of compounds, other methods proved more effective in terms of cost and time [41]. Fernandez-Gonzalez et al. applied PHWE followed by SPME to the determination of PAHs in marine sediment [42]. The authors took advantage of the suitability of the PWHE extract to process further by SPME to obtain a method with improved limits of detection (LODs) (0.4 15 lg/kg) and intrinsic clean up of the extract. 2.1.9. Ionic liquids as green extraction solvents. Roomtemperature ILs have emerged as a new type of green solvent, generally comprising quaternary nitrogen cations (e.g., alkylammonium, dialkylimidazolium, and alkylpyridinium) and the appropriate anion. They have attracted increasing interest in the chemistry community because of their remarkable, uncommon properties (e.g., negligible vapor pressure, non-ammability, high thermal stability, and tunable viscosity and miscibility with water and organic solvents) [31,34]. ILs have been employed in liquid-liquid extraction, namely DLLME, stationary phases for GC and supported liquid-membrane extraction, and in chemical synthesis, catalysis and electrochemistry [43].
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Liu et al. published an excellent review on the application of ILs in analytical chemistry [44], updated in 2009 with a list of imidazolium and non-imidazoliumbased ILs, their characteristics and the main applications in sample preparation. The problems and challenges in this area were also covered. Zhou and co-workers proposed an interesting application of ILs called temperature-controlled IL DLLME. 1-Hexyl-3-methylimidazolium hexauorophosphate [C6MIM][PF6] was used as the extraction solvent for the enrichment of pyrethroid pesticides from water. At 70C, the IL was totally soluble in the water sample and extracted the pesticides, then the solution was cooled, became turbid and the IL was recovered by centrifugation [36]. The same IL was suitable to extract directly four heterocyclic pesticides from water without any heating and cooling, so resulting in a faster procedure suitable for thermolabile compounds [36]. ILs have been regarded with great hope in order to enlarge the range of applicability of DLLME, desirably to more polar compounds.
3. Greening separation and detection techniques The separation step prior to detection of the analytes is crucial for the majority of complex samples. Recent advances in separation techniques have decreased the use of solvents, time taken and costs, making them greener. Regarding the detection techniques, they are quite green because limited quantities of hazardous chemicals are used. We therefore next discuss some recent improvements in separation and detection techniques (nonbiological and biological approaches) to convert them into greener techniques. 3.1. Biological approaches The main advantages of biological approaches are: rapid analysis; sample pre-treatment is simple or not required; possibility of in situ analysis; low volumes of sample required; and, use of solvents low or not required. 3.1.1. Immunochemical techniques. The binding properties of an antibody (Ab) to an antigen (Ag) have been used for the development of a wide variety of analytical techniques applicable in rapid environmental control for the measurement of both single and multiple analytes [4448]. Immunoassays offer a number of advantages in green environmental analysis over conventional methods, because they can provide fast, simple and cost-effective detection, with sensitivity in most of cases comparable to conventional techniques and requiring no or minimal quantities of solvent, with minimal sample pre-treatment required (so reducing waste) and reduced energy consumption during sample analysis. In addition, more advanced formats (e.g., immunoassays) can be designed to
operate on-line in the eld. The main limitations encountered with these techniques are sometimes poor thermal and chemical stability of immunoreagents, cross reactivity between structurally-related compounds, and possible matrix effects. Current development is therefore focused on the application of new materials in order to improve the stability and the specicity of immunoreagents. At present, most immunochemical advances in environmental analysis have been carried out using enzymebased immunoassays (EIAs), especially in immunosensing, which we review in the next section devoted to biosensors. EIAs typically use a change in color, emission of light, or other signal to measure the concentration of an analyte. EIAs are based on enzymes to enhance the signal, and they are widely used for rapid environmental analysis, especially those based on heterogeneous conditions [e.g., enzyme-linked immunosorbent assays (ELISAs)]. The main enzymes used are horseradish peroxidase (HRP), alkaline phosphase (AP) and b-galactosidase. In recent years, a great effort of development has been made to develop ELISAs for emerging pollutants {e.g., surfactants [49,50]} or antibacterial agents {e.g., triclosan [51,52]}. For screening purposes, different immunoassays using dipstick formats have been developed [53]. Cho et al. [54] developed a direct competitive ELISA for fenthion in microtiter-plate and dipstick formats. The microtiterplate ELISA showed an IC50 value of 1.2 lg/L with an LOD of 0.1 lg/L. The Abs showed negligible crossreactivity with other organophosphorous pesticides. The use of the dipstick format using a support membrane allowed the quick visual detection of fenthion in concentrations >10 lg/L. The IC50 value of the dipstick format using reectance detection was 15 lg/L with an LOD of 0.5 lg/L. The recoveries of fenthion from spiked vegetable samples using the two formats without any prior enrichment or clean-up steps were 87-116%. Recently, Lisa et al. [55] developed a gold-nanoparticle (GNP)-based dipstick competitive immunoassay to detect organochlorine pesticide, such as DDT, at ng level. GNPs of denite size were synthesized and conjugated to anti-DDT Abs. The intensity of color development was inversely proportional to the DDT concentration. The lowest LOD of DDT was 27 ng/mL under optimal conditions. Screening assays based on dipstick format are a good example of rapid, cost-effective screening methods, with reduced time of analysis, waste and solvents. 3.1.2. Biosensors. A biosensor is dened by IUPAC as a self-contained integrated device that is capable of providing specic quantitative or semi-quantitative analytical information using a biological-recognition element (biochemical receptor), which is retained in direct spatial contact with a transduction element. Fig. 1
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shows a general scheme of biosensor technologies.Biosensors offer capabilities for rapid, miniaturized, on-line and at-site analysis, with minimal waste production and energy cost. Several reviews on biosensors and biological techniques for environmental analysis have been published in recent years [5660]. Biosensors comprise two main parts: the physicochemical transduction element (e.g., electrochemical, optical, mass-sensitive, and thermal); and, the biological receptor (e.g., enzymes, Abs, DNA and whole cells). 3.1.2.1. Electrochemical transduction. Typically in (bio-) electrochemistry, the reaction under investigation would either generate a measurable current (amperometric), a measurable potential or charge accumulation (potentiometric) or measurably alter the conductive properties of a medium (conductometric) between electrodes. Other types of electrochemical-detection technique (e.g., impedimetric, which measures impedance, both resistance and reactance, and eld-effect, which uses transistor technology to measure current as a result of a potentiometric effect at a gate electrode) have also been reported. However, most applications for environmental analysis have been based on amperometric and potentiometric congurations. Electrochemical biosensors do not suffer the drawback of high sensor set-up complexity and cost. Because of their simplicity, they have given rise to a great variety of low-cost devices based on different formats [61,62]. This is due to their close link to developments in low-cost production of microelectronic circuits and their easy interface with normal electronic read-out and processing. Other inherent advantages are their robustness and easy miniaturization. However, several aspects could be considered to have held back the emergence of additional breakthrough applications built on electrochemical biosensing. Electrochemical biosensors have suffered from a
lack of surface architectures allowing high enough sensitivity and unique identication of the response with the desired biochemical event. For example, pH and ionic strength in biouids can differ signicantly, which affects the response of important classes of biosensors (e.g., immunosensors), so there has recently been increased emphasis on using nanotechnology to shrink the dimensions of electrochemical-sensor elements to sizes that can increase the signal-to-noise ratio for processes designed to occur at the interface of the device and to nd ways of using, e.g., multiple enzymatic labels to increase the signal per event. In biosensing, the measurement of electrical properties to extract information from biological systems is normally electrochemical in nature, whereby a bioelectrochemical component serves as the main transduction element. Although biosensing devices employ a variety of recognition elements, electrochemical-detection techniques predominantly use enzymes, mostly due to their specic binding capabilities and biocatalytic activity. Enzyme electrodes have the longest tradition in the eld of biosensors [63]. Table 1 shows some examples of recent environmental applications of electrochemical-enzyme biosensors. In recent years, the huge increased interest in nanomaterials was driven by their many desirable properties. In particular, the ability to tailor the size and the structure and hence the properties of nanomaterials offers excellent prospects for designing novel sensing systems and enhancing the performance of the bioanalytical assay. An important challenge in amperometric enzyme electrodes is the establishment of satisfactory electrical communication between the active site of the enzyme and the electrode surface [64,65]. A wide range of enzyme electrodes based on dehydrogenase or oxidase enzymes rely on amperometric monitoring of the liberated NADH or hydrogen peroxide. The anodic detection of these species is often hampered by the large over voltage encountered for their oxidation.
Figure 1. General scheme of biosensors.
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Table 1. Examples of electrochemical biosensors reported for environmental analysis Analyte/Effects Bisphenol A Carbamates Catechol, p-cresol, phenol, p-chlorophenol, and p-methylcatechol Fenitrothion and ethyl p-nitrophenol thiobenzenephosphonate Genotoxicity Heavy metals Biological receptor/Transducer Tyrosinase/Amperometric detection AChE, BChE/Potentiometric detection Tyrosinase/Amperometric detection Matrix Drinking and surface water Aqueous synthetic samples Spring and surface water Limit of detection 0.02 lM 1.5.1052.5.103 M 9 108 M Ref. [122] [123] [124]
Whole-cell biosensor/Amperometric detection DNA/Amperometric detection DNA/Amperometric detection
Water
1.4 lg/L of fenitrothion and 1.6 lg/L of EPN
[82]
Wastewater Water 4.0 1011, 1.0 1010, 1.0 109, and 5.0 109 M for Cu(II), Pb(II), Cd(II), and Fe(III), respectively 1.0 1011 M 0.25 mg/L
[73] [125] [75]
PAT gene Surfactants
DNA/Impedance measurement Bacteria/Amperometric detection
Water Water
[126] [127]
It has been proved that the redox activity of hydrogen peroxide [66] and NADH [67] is enhanced using CNTmodied electrodes, which help to overcome voltage limitations. Gong et al. [68], reported a nanosized calcium carbonatechitosan (nanoCaCO3chi) composite lm possessing a three-dimensional (3D) porous, network-like structure, which provided a favorable, biocompatible microenvironment to immobilize enzymes. By using such a composite lm as an enzyme-immobilization matrix, a highly sensitive, stable acetylcholinesterase (AChE) sensor was achieved for determination of methyl parathion as a model of organophosphatepesticide compounds. The inhibition of methyl parathion was proportional to its concentration, and the LOD was found to be as low as 1 ng/mL. Recently, an electrochemical biosensor for the determination of organophosphorous insecticides in vegetable
crops, was described. The self-assembled monolayers (SAMs) of SWCNTs wrapped by thiol-terminated singlestrand oligonucleotide (ssDNA) on gold was utilized to prepare nanosized polyaniline matrix for AChE-enzyme immobilization [69]. The LOD of the biosensor for methyl parathion and chlorpyrifos pesticides was found to be 1 1012 M in both cases. Electrochemical immunosensors have been used for environmental analysis in amperometric, potentiometric, and conductimetric congurations. Table 2 gives different examples of immunosensors. For example, an amperometric immunosensor for ricin in water by using graphite and CNT-paste electrodes was described by Suresh et al. [70]. Due to their wide range of physical, chemical and biological properties, nucleic acids (NAs) have been incorporated into a wide range of biosensors and bio-
Table 2. Immunosensors for environmental applications Analytes 2,4-Dichlorophenoxyacetic acid 2,4-Dichlorophenoxyacetic acid Atrazine Atrazine Atrazine Chlorpyrifos Dioxins Hormones Isoproturon Oganophosphorous and carbamate Pesticides Pesticides Stanozolol Transducer SPR Fluorescence/Chemiluminescence SPR SPR Impedimetric SPR TIRF Fluorescence QCM TIRF Electromagneto-immunosensor LSPR Matrix Water Water River water Water Rain, surface, marine and ground water Surface, ground and drinking water Fly ash Water Water Water Water Water Water Limit of detection 0.1 ng/mL 0.02 ng/mL 20 ng/L 1 lg/mL 20 ng/L 55 ng/L 0.1-0.01 ng/mL 1.4 ng/L 9.7 ng/L 1 ng/mL Ref. [128] [129] [94] [130] [131] [132] [133] [134] [135] [136] [134] [137] [103]
QCM, Quartz-crystal microbalance; SPR, Surface-plasmon resonance; LSPR, Localized surface-plasmon resonance; TIRF, Total internal reection.
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analytical assays, many of which possess interesting features for environmental applications. Several DNA biosensors and bioassays have been reported for the detection of chemically-induced DNA damage. The structure of DNA is very sensitive to the inuence of environmental pollutants. Different groups of organic pollutants are characterized by a great afnity to DNA, causing mutagenesis and carcinogenesis, so it is very convenient to use DNA-containing systems {e.g., DNA-based biosensors [71]} to perform genotoxic assays or for rapid testing of pollutants for mutagenic and carcinogenic activity. The determination of genotoxic compounds was measured by their effect on the oxidation signal of the guanine peak of calf-thymus DNA immobilized on the surface of disposable electrodes and investigated by chronopotentiometric analysis by Mascinis group [72]. However, the rapid screening of genotoxic compounds using the molecular interaction between surface-linked DNA and the target pollutants or drugs has been applied in many different congurations using electrochemical, optical and mass-sensitive transducers [7375]. There is also an ongoing effort in the area of biosensor technology for measuring NA hybridization. In this case, a sequence-specic probe (usually a short synthetic oligonucleotide) is integrated within a signal transducer. The probe, immobilized onto the transducer surface, acts as the biorecognition molecule and recognizes the target DNA or RNA. Optical, electrochemical and mass detectors are mainly used for transducing the biorecognition event into an analytical signal. However, such NA-sensing applications require high sensitivity, and substantial efforts have been devoted to amplication of the transduction of the oligonucleotide interaction. NPbased amplication schemes have led to improved sensitivity of bioelectronic assays by several orders of magnitude. Several amplication processes can be used for dramatically enhancing the sensitivity of NP-based bioelectronic assays. The use of inorganic-nanocrystal tracers for a multitarget electronic detection of DNA [76] can be scaled up and multiplexed. One-dimensional nanowires can also be used for bridging two closely-spaced electrodes for label-free DNA detection. For example, a p-type silicon nanowirefunctionalized with peptide NA (PNA) probes was shown to be extremely useful for real-time, label-free, conductimetric monitoring of the hybridization event [77]. This relies on the binding of the negatively-charged DNA target that leads to an increase in conductance, reecting the increased surface charge. CNTs can also lead to ultrasensitive bioelectronic detection of DNA hybridization. For example, CNTs can be used as carriers for several thousand enzyme tags and for accumulating the a-naphthol product of the enzymatic reaction. Such a CNT-derived double-step 1354
amplication pathway (of both recognition and transduction events) allows detection of DNA down to the 1.3 zmol level. Recently, Feng et al. [78] reported a GNP/polyaniline NT membrane on a glassy carbon electrode (Au/nanoPAN/GCE) for DNA-impedance sensing. The synergistic effect of the two kinds of nanomaterials, nanogold and nanoPAN, could dramatically enhance the sensitivity for the DNA-hybridization recognition. The highly sensitive, sequence-specic detection of single-stranded oligonucleotide using non-oxidized silicon nanowires (SiNWs) was demostrated by Zhang et al. [79]. To maximize device sensitivity, the surface of the SiNWs was functionalized with a densely-packed organic monolayer via hydrosilylation, and subsequently immobilized with PNA capable of recognizing the label-free complementary target DNA. Because of the selective functionalization of the SiNWs, binding competition between the nanowire and the underlying oxide was avoided. Thus, as reported above, the promising performance of NA biosensors for hybridization studies has been demonstrated in several studies. Several microbial biosensors for ecotoxicity assessment have been reported using electrochemical congurations. An example of a recent development is a biological oxygen demand (BOD) biosensor using salt-tolerant Bacillus licheniformis for their application in sea water [80]. In another recent work, a novel reactor-type biosensor for rapid measurement of BOD was developed, based on using immobilized microbial cell (IMC) beads as recognition bio-elements in a completely mixed reactor that was used as determining chamber, replacing the traditional membrane used as recognition bio-element. This novel kind of BOD biosensor signicantly increased the sensitivity of the response and the precision of detection, and prolonged the life of the recognition element [81]. In addition to BOD determination, bacterial biosensors have been developed to detect different groups of organic pollutants. For example, amperometric biosensors based on genetically engineered Moraxella sp. and Pseudomonas putida with surface-expressed organophosphorus hydrolase (OPH), were developed by Lei at al. [82] for the detection of organophosphorus pesticides. Under optimal operating conditions, the biosensor was able to measure as low as 277 ppb of fenitrothion. In another example, a bacterial biosensor using screen-printed electrodes with E. coli was reported for wastewater-toxicity assessment et al. [83]. by Farre 3.2. Optical transducers Optical transducers are based on various technologies of optical phenomenon (e.g., adsorption, uorescence, phosphorescence, polarization, rotation, interference) or non-linear phenomena (e.g., second harmonic generation). The choice of a particular optical method depends
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on the nature of the application and desired sensitivities. In practice, ber optics can be coupled with all optical techniques, thus increasing their versatility. The opticalbiosensor formats may involve direct detection of the analyte of interest or indirect detection through optically-labeled probes. 3.2.1. Evanescent wave. Evanescent wave is produced in the external media (refractive index) of a waveguide by the electromagnetic eld associated to the light guided by total internal refection (TIR). The electromagnetic eld does not abruptly switch to zero at the interface between the two media, but decays exponentially with distance from the interface. The penetration depth of the evanescent eld is dened as the distance where its strength is reduced to 1/e of its value at the interface and generally has a value of $100 nm. The penetration depth depends on the incident angle at the interface and is proportional to the wavelength of the excitation light. When molecules with an absorption spectrum that includes the excitation wavelength are located in the evanescent eld, they absorb energy, leading to attenuation in the reected light of the wavelength. With the aim of improving detectability, different biosensors have been developed, combining this principle with labeled molecules that are able to re-emit the absorbed evanescent photons as uorescence at a longer wavelength. Part of this emission is coupled back to the waveguide and in this way is transmitted to the receptor. This phenomenon is known as total internal reection uorescence (TIRF), which has been used with planar and ber-optic waveguides as signal transducers in a number of biosensors. In these devices, light is propagated down a waveguide, which generates an electromagnetic wave (evanescent wave) at the surface of the optically denser medium of the waveguide and the adjacent less optically dense medium. The amplitude of the standing wave decreases exponentially with distance into the lower refractive index material. The uorescence of a uorophore excited within the evanescent eld can be collected either outside the waveguide, or by coupling the emission frequencies back into the waveguide. The biological sensing element is immobilized on the side rather than at the end of the waveguide. Based on the evanescent wave transducing principle, atrazine was detected at concentrations around 0.1 lg/L [84] and cyclodiene insecticides in the lg/L range [85]. TIRF is a fast, non-destructive, sensitive and versatile technique that is well suited for monitoring biomolecular interactions. TIRF allows monitoring conformational changes, orientation changes, and lateral mobility of biomolecules. TIRF has been used in a large number of biosensors. Examples of application are the river analyzer (RIANA) and the automated water analyzer computer supported system (AWACSS) [86,87]. Hormones, pesticides, antibiotics, and endocrine-disrupting chemi-
cals are among the organic pollutants that have been detected with this biosensor system. 3.2.2. Optical waveguide light-mode spectroscopy. Optical waveguide light-mode spectroscopy (OWLS) is another sensing technique using evanescent eld for the in situ, label-free study of surface processes at molecular levels. It is based on the measurement of the resonance angle of linearly polarized laser light, diffracted by a grating and coupled into a thin waveguide layer. Incoupling is a resonance phenomenon that occurs at a dened angle of incidence that depends on the refractive index of the medium covering the surface of the waveguide. In the waveguide layer, light is guided by TIR to the edges, where it is detected by photodiodes. By varying the angle of incidence of the light, the spectrum obtained allows effective refractive indexes to be calculated for both electrical and magnetic eld waves [47]. For example, a label-free carp-vitellogenin sensor was reported for its strong potential for on-site monitoring on the possible contamination of edible sh with endocrine disruptors as a sum parameter in an inland carp farm, with an LOD for vitellogenin of 0.00675 nM, [88]. 3.2.3. Surface-plasmon wave. A surface-plasmon wave can be described as a light-induced collective oscillation in electron density at the interface between a metal and a dielectric. At surface-plasmon resonance (SPR), most incident photons are absorbed or scattered at the metal/dielectric interface, so reected light is greatly attenuated. The resonance wavelength and angle of incidence depend upon the permittivity of the metal and dielectric. SPR biosensors are based on the special electromagnetic wave-surface plasmon-polariton-toprobe interactions between an analyte in solution and a biomolecular-recognition element immobilized on the SPR-sensor surface. This principle has been extensively investigated for monitoring of biological interaction in different types of congurations. Since distinct SPR prototypes have appeared in the market (e.g., Biacore, IASys, and Sensia), a signicant number of applications of this principle have been reported in recent years [89 93]. Several papers have reported the analysis of pesticides in environmental samples with minimal or no sample preparation [94]. In another example, an assay for bioeffect-related screening of chemicals with thyroid-disrupting activity was developed. In this assay, two thyroid-transport proteins, thyroxine-binding globulin and recombinant transtheryn, were applied in an inhibition-assay format using a Biacore 3000 with CM5 biosensor chips coated with 1-thyroxine [95]. An SPR immunosensor was developed for the detection of 2,4-dinitrophenol at ultra-low concentrations (immunoassay range 1 ppt1 ppb). The sensor strategy
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was based on competitive immunoreaction between 2,4dinitrophenol and a 2,4-dinitrophenol-protein conjugate, namely DNP-bovine serum albumin conjugate (DNP-BSA). Anti-2,4-dinitrophenol monoclonal Ab was immobilized on a gold thin-lm-coated SPR-sensor chip by chemical coupling [96]. SPR biosensors using nuclear receptors have been the basis of several biosensors for environmental analysis. For example, the estrogen receptor (ER) was the basis to assess the estrogenic potency of complex matrices polluted with endocrine-disruptor compounds (EDCs). Steroid hormones induce different effects in mammalian cells after binding to specic intercellular receptors, which are ligand-dependent transcription factors. Many endocrine disruptors can bind to estrogen receptors as agonists or antagonists, so the binding ability of the chemicals towards the ER is used to test their potential environmental toxicity. The advantage of receptor assays is that they are quite simple to perform and they allow identication of all endocrine disrupters that act through the estrogen receptor. The natural sensing element most commonly used is the human ER. Using SPR, [97] Usami et al. developed a simple competitive assay for the evaluation of different chemicals using human recombinant ER. The system measured the binding between estradiol immobilized on the sensor chip and an injected human recombinant ER. In another example using an optical SPR sensor and human estrogen a receptor, Hock et al.[98] performed binding studies with estradiol and xenoestrogens. Butala et al. [99] described the use of a fractal analysis to model the binding and dissociation kinetics between analytes in solution and ER immobilized on an SPR-sensor chip. An evanescent wave-based biosensor [100] was developed with laser-based ber optics using uorescent dye-labeled recombinant human estrogen receptor-alpha (rhERalpha) and hERbeta as probes. A three-tiered approach evaluating various steps in the formation of the estrogen-receptor complex and its subsequent activity was developed for instrument calibration to detect estrogen mimics in biological samples, water and soil. Using this approach, binding afnities and activities of certain known estrogen mimics were determined for their use as calibrator molecules. Results indicated rhERalpha and rhERbeta may be employed as probes to distinguish estrogen mimics with a broad range of afnities. The development of large-scale biosensor arrays comprising highly miniaturized signal-transducer elements that enable real-time, parallel monitoring of multiple species is an important driving force in biosensor research. This is particularly signicant in high-throughput screening applications, where many thousands of ligand-receptor or protein-protein interactions must be rapidly examined.
3.2.4. Localized SPR. Recently, several research groups have begun to explore other strategies for the development of optical biosensors, based on the extraordinary optical properties of noble-metal NPs, which exhibit a strong UV-vis absorption band that is not present in the spectrum of the bulk metal. This absorption band results when the incident photon frequency is resonant with the collective oscillation of the conduction electrons and is known as Localized SPR (LSPR). LSPR excitation results in wavelength-selective absorption with extremely large molar extinction coefcients of $3x1011/M/cm, resonant Rayleigh scattering with an efciency equivalent to that of 106 uorophors, and enhanced local electromagnetic elds near the surface of the NP, which are responsible for the intense signals observed in all surface-enhanced spectroscopies. It is well established that the peak-extinction wavelength, kmax, of the LSPR spectrum depends upon the size, the shape, and the interparticle spacing of the NP as well as its dielectric properties and those of the local environment. Consequently, there are at least four different NP-based sensing mechanisms that enable transduction of macromolecular or chemical-binding events into optical signals based on changes in the LSPR extinction or scattering intensity, shifts in LSPR kmax, or both. These mechanisms are: (1) resonant Rayleigh scattering from NP labels in a manner analogous to uorescent dye labels; (2) NP aggregation; (3) charge-transfer interactions at NP surfaces; and, (4) local refractive index changes. It has been demonstrated that nanoscale biosensors can be realized through shifts in the LSPR kmax of triangular silver NPs [101,102]. These wavelength shifts are caused by adsorbate-induced local refractive-index changes in competition with charge-transfer interactions at the NP surface. Triangular silver NPs have been shown to be unexpectedly sensitive to NP size, shape, and local dielectric environment [103]. Recently, the current state of development of SPRimaging (SPRi) technology and its application, including commercially available SPRi instruments, was reviewed by Scarano et al. [104]. Different examples of the application of biosensors based on bioluminescent bacteria for wastewater monitoring have been reported. The construction of wholecell, genetically-modied, bioluminescent biosensors and their immobilization on thin lms of poly(vinyl alcohol) cryogels was carried out by Philp et al. [105. The biosensor was designed for use in monitoring the toxicity of industrial wastewaters containing phenolic materials. Several recombinant bioluminescent bacteria have been employed to set up a multi-channel, continuous, watertoxicity-monitoring system. That developed by the research group of Kim was based on channels, each
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hosting a different recombinant bacterial strain, and comprised two mini-bioreactors to enable continuous operation (i.e. without system interruption due to highly toxic samples) [106]. A uorescence multiple-strain algal biosensor was constructed for the detection of herbicides inhibiting photosynthesis. In this work [107], nine different microalgal strains were immobilized on an array biochip using permeable membranes. The biosensor allowed online measurements of aqueous solutions passing through a ow cell using chlorophyll uorescence as the biosensor-response signal. Herbicides atrazine, simazine, diuron, isoproturon and paraquat were detectable within minutes. A recent work [108] presented the development of an optical biosensor for environmental monitoring based on computational and biotechnological tools for engineering the photosynthetic D1 protein of Chlamydomonas reinhardtii. The uorescence properties were exploited and a reusable, portable, multi-array optical biosensor for environmental monitoring was developed with LODs of 0.0083.0 109 M for triazine herbicides. 3.3. Mass-sensitive sensors Measurement of small changes in mass is a form of transduction that has been used for biosensor development. Piezoelectric and surface-acoustic-wave devices can be grouped under this category (Fig. 1). Although this principle has been less reported for environmental applications, its capabilities (e.g., the possibility of miniaturization and the high sensitivity and specicity achieved when coupled to the appropriate bioreceptor) make it one of the most promising approaches. The vibration of piezoelectric crystals produces an oscillating electric eld, in which the resonant frequency of the crystal depends on its chemical nature, size, shape and mass. These crystals can be made to vibrate at a specic frequency of oscillation, which depends on the electrical frequency. The frequency of oscillation therefore depends on the electrical frequency applied to the crystal as well as the crystal mass. When the mass increases due to the binding of analytes, the oscillation frequency of the crystal changes and this change can be measured. The general equation of crystal microbalances can be summarized as follows when the change in mass (m) is very small compared to the total mass of the crystal: Df Cf 2 Dm=A where f is the vibration frequency of the crystal in the circuit, A is the area of the electrode and C is a constant determined in part by crystal material and thickness. Piezoelectric crystals, sometimes referred to as quartz crystal microbalances (QCMs), are typically made of quartz and operate at frequencies of 110 MHz. These
devices can operate in liquids with a frequency-determination limit of 0.1 Hz; the LOD of mass bound to the electrode surface is about 110 1011 g. Acoustic wave devices, made of piezoelectric materials, are the most common sensors, which bend when a voltage is applied to the crystal. Acoustic wave sensors are operated by applying an oscillating voltage at the resonant frequency of the crystal, and measuring the change in resonant frequency when the target analyte interacts with the sensing surface. 3.4. Non-biological techniques 3.4.1. Separation techniques: greening liquid chromatography. High-performance LC (HPLC) and GC are often used prior to the detection systems for organic-pollutant analysis. GC is a separation technique that, as the name suggests, does not use solvents but relies on the separation of the analytes in the gas phase. However, HPLC uses large amounts of solvents, and it gained popularity and became one of the preferred techniques for analyzing polar contaminants. HPLC has been used in laboratories worldwide over the past 30 years [109,110]. However, advances in HPLC have led to increased interest in comparing the ultimate performance limits of methodologies aimed at increasing the resolving power per unit time (e.g., use of nanoows for HPLC) [111]. However, it is not commonly used in the environmental analysis. Recently, ultra-high-pressure LC (UHPLC) was introduced to enhance sample throughput and reduce analysis time and ultimately mobile-phase consumption compared to HPLC. UHPLC takes advantage of HPLC columns packed with sub-2 lm particles, implying operating at high backpressures, to reduce analytical run times signicantly. By decreasing the particle size of the packing material, the analyst can reduce the height of a theoretical plate, making possible shorter column lengths and widening the range of usable ow rates without sacricing separation power. Consequently, the analytical method is faster without losing separation quality [112]. Also, in chromatography, increasing the temperature of the mobile phase above ambient temperature reduces the time to obtain the chromatogram and consequently reduce solvent use [113]. This kind of application is called high-temperature LC (HTLC) and can be a great value, since the concomitant decrease in mobile-phase viscosity and increase in solute diffusivity with temperature allow high ow rates that lead to short analysis time without loss of efciency or signicant increase in column back-pressure [113]. As the dielectric constant and surface tension of water decrease with increasing temperature, a large proportion of organic solvent is replaced by water in the mobile phase [113]. If the temperature of the mobile phase (water) increases 5C, it is comparable to the increase of 1% in acetonitrile [114],
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and, a 1% reduction of methanol can be obtained when the temperature of the mobile phase increases 3.75C [115]. Water above its boiling point at atmospheric pressure to its supercritical temperature therefore exhibits reduced viscosity, increasing its capability to dissolve non-polar compounds and decrease polarity [116]. Several authors have studied the use of heated water in HPLC separations [115], but the chromatographic behavior using UHPLC columns (HT-UHPLC) is relatively new [113]. Nguyen et al [113] evaluated the combination of high temperature and UHPLC for the rst time for the separation of several doping agents and a cocktail of pharmaceuticals. Both separations were obtained in less than 1 min at 90C and at pressures above 400 bar. In addition, a shorter column (30 mm) was used at high temperature and allowed a separation of eight pharmaceuticals in only 40 s [115]. Two chromatographic methods (with UHPLC Aquity columns 10 cm and 30 cm long) for the separation of eight drugs were compared at 60C and 180C [115]. The 30-cm column was obtained by linking 3 10 cm columns together in series using zero-dead-volume ttings in order to maximize the separation. The authors calculated the ow rate with an experimental model developed based on data obtained using a range of model drugs, which demonstrated the relationship between temperature, ow and pressure. For the chromatographic separation of the target analytes, they determined that, at a temperature of 60C, the maximum ow rate for a back-pressure of 15,000 psi was 0.4 mL/min, and, at temperature of 180C, the ow rate was calculated to be 1 mL/min. It was shown that there was an improvement in efciency using the longer columns, and the retention time was dramatically reduced by using the higher temperature. The estimated back-pressure of the 30-cm column at room temperature was 65,000 psi, so it was impractical with any commercially-available chromatographic system [116]. Another green chromatographic technique for the separation of organic pollutants is planar chromatography, particularly high-performance thin-layer chromatography (HPTLC). This technique is simpler and more cost effective, and requires less solvent than the HPLC for the chromatographic separation of a wide spectrum of substances [117]. However, due to the advances in HPLC, HPTLC has taken a back seat, in spite of its multiple advantages (e.g., the possibility of performing parallel analysis, high exibility towards chromatography and detection, and tolerance of samples with highly complex matrices). This technique is a well-known offline approach to separation, but it is underestimated. Some applications showed that HPTLC can be superior to HPLC {e.g., the rapid analysis of bioactive secondary metabolites in marine sponges by coupling HPTLC with bioluminescence and mass spectrometry (MS) showed 1358
the utility of this technique [117]. HPLTC presented a very fast response and the organic mobile phase was evaporated from the plate, so the solvent could not impede direct detection with the bioassay. If separated compounds were bioactive, the inhibited bacterial luminescence could be identied as dark zones on the luminescence background. Microbiological detection revealed new compounds. The mass spectra of the bioactive zones were recorded within seconds by direct analysis in real time (DART) or with an exact mass analyzer LTQ Orbitrap XL hybrid Fourier transform (FT)MS, allowing identication of avarone as a bioactive metabolite of the Dysidea avara sponge. This methodology proved to be very effective for not only the detection but also the identication of unknown bioactive metabolites in sponges [117]. Another feature of the technique is the novel in situ pre- and post-chromatographic derivatization of the target analytes. For example, derivatization of the metabolites from the Dysidea avara sponge by dipping the plate into sulfuric acid, dissolving in water (to cool) and then in methanol, took 1 min [117]. Another example is the rapid, sensitive determination of acrylamide in drinking water by HPTLC with uorescence detection after derivatization with dansulnic acid [118]. After SPE, aliquots of 100 lL of water extracts were sprayed onto an HPTLC silica-gel plate at the starting and covered in situ with derivatization agent dansulnic acid by heating the plate for different periods. Subsequently, the products containing uorescent chromophores were analyzed with uorescent detection [118]. Coupling HPTLC with UV or MS detectors, the quantication of target analytes in the plate is also feasible. Aranda and Morlock [119] developed a method for the quantication of caffeine in pharmaceutical and energydrink samples using stable-isotope dilution. After sample preparation, samples and caffeine standard were applied on silica-gel 60 F254 HPTLC plates and spotted, with caffeine-d3 used for the correction of the plunger positioning. This work showed that isotope dilution was a good option for correcting the plunger positioning because the stable-isotope internal standard showed the same chromatographic behavior (same hRf value) as the analyte and thus the positioning error could be nullied. The calibration showed a linear regression with a determination coefcient of 0.9998 [119]. 3.4.2. Detection techniques. In spectroscopic and electrochemistry techniques, the size of the sample is relatively small and there is no sample-preparation step. These kinds of technique are fundamentally green. However, detection techniques (e.g., MS) need sample preparation and frequently chromatographic separation of the sample prior to detection. Developments in MS systems have almost exclusively been to improve atmospheric pressure ionization (API) interfaces with
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systems where the samples are introduced in a liquid stream, namely atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). Some efforts have been made to reduce the amount of solvent introduced into the ion source of the electrospray. Developments in the sub-mL/min ow range are referred to as nanoelectrospray. However, the new ion-source developments, many of which deal with surface analysis and introduction of solid samples, eliminate the use of solvents because no sample preparation is needed and no solvent is used to introduce sample into the MS system. All the traditional API sources {e.g., ESI, matrix-assisted laser desorption ionization (MALDI), APCI or atmospheric pressure photoionization (APPI)} still require extensive sample-preparation steps before the sample can be introduced into the MS instruments. With the introduction of desorption ESI (DESI) and DART, it became possible, for the rst time, to analyze samples directly in their native condition without sample-preparation steps [120]. However, although these techniques are relatively new, they have been applied successfully for the detection of organic pollutants in the environment. The determination of saturated non-functionalized
Table 3. Main green analytical approaches and their advantages
hydrocarbons in petroleum distillates derived from crude oils was performed by DESI. These compounds are difcult to analyze by MS using API methods. In the DESI method, the hydrocarbons were oxidized in situ by initiating an electrical discharge during DESI to generate the corresponding alcohols and ketones [121]. This kind of reactive DESI experiment can be utilized as an in situ derivatization method for rapid, direct analysis of alkanes at atmospheric pressure without sample preparation. The LOD for alkanes from pure samples was 20 ng and the technique was successfully implemented using exact-mass measurements. 4. Future trends Table 3 presents a scheme of the main GAC techniques and their advantages. The application of GAC methods is desirable from the point of view of the environment, human health and the economy. Progress in analytical methodologies has contributed to the development of new, greener options, involving smaller amounts of toxic reagents, reducing waste and saving energy.
Instrumental techniques Step Sample preparation Type of technique Solvent-reduced Techniques Solvent-reduced solid-phase extraction: Solid-phase micro extraction (SPME), Stir-bar sorptive extraction (SBSE), Microextraction in packed syringe (MEPS), Thin-lm microextraction (TFME) Liquid-liquid microextraction: Single-drop microextraction (SDME), Organic solvent lm (OSF), Continuous-ow microextraction (CFME), Hollow-ber liquidphase microextraction (HFLPME), Dispersive liquid-liquid microextraction (DLLME), Supercritical uid extraction (SFE), Subcritical water extraction (SWE) also known as pressurized hot-water extraction (PHWE), Ionic liquids as green extraction solvents Ultra-high-pressure liquid chromatography (UHPLC) High-performance thin-layer chromatography (HPTLC). Nanoelectrospray Desorption electrospray (DESI) and direct analysis in real time (DART) Advantages Reduction of solvent consumption, improving and simplifying sample pretreatment. In some cases, toxic solvents are substituted by water as in SWE; in other cases, they are substituted by new types of green solvent generally comprising quaternary nitrogen cations (e.g., alkylammonium, dialkylimidazolium, and alkylpyridinium) and the appropriate anion, as in ionic liquids
Separation
Greening Liquid Chromatography
Detection Techniques
Mass Spectrometry
Signicant reduction of analytical run times, consequently reducing solvent consumption Simple and cost effective with reduced solvent consumption Solvent reduction in the electrospray source Elimination of sample preparation. It is possible to analyze samples directly in their native conditions without sample preparation
Biological techniques Immunochemical Enzyme-linked immunosorbent assays (ELISAs) Biosensors
It can provide fast, simple and cost effective detection, with sensitivity in most of cases comparable to conventional techniques requiring none or minimal quantities of solvent, with minimal sample pretreatment requisites (reducing wastes) and a reduced energy consumption during sample analysis. In addition, more advanced formats (e.g., immunoassays) can be designed to operate on-line in the eld and or to avoid sample pretreatment. Biosensor offer capabilities for rapid, miniaturized, on-line and at-site analysis, with minimal waste production and costs of energy.
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Regarding extraction and purication steps, microextraction techniques represent a step forward for the development of environmentally-friendly analytical techniques. The range of potential applications of these techniques is in constant development {e.g., in the near future, automated SPME will be extended to more polar analytes (e.g., pharmaceutical compounds)}. However, miniaturization of analytical systems, in line with the concept of micro total analysis systems (lTAS), is still underdeveloped. In chromatographic separations, a lot of efforts have been made to reduce time and organic solvents (e.g., UHPLC or the reintroduced HPLTC). These techniques minimize the use of solvents in performing rapid separations. Other options (e.g., on-line SPE) have gained interest in this sense. Several approaches have also recently emerged for eliminating the use of organic solvents in ionization techniques for MS. For example, ambient DESI techniques, in a single operational step, successfully analyze directly the samples in their native form without the use of solvents for sample preparation and introduction to MS. Finally, more advanced analytical technologies (e.g., biosensors) are generally greener techniques, because low volumes of samples and solvents are involved, and, in many cases, sample pre-treatment can be avoided. However, there are still few of these techniques implemented in routine environmental analysis, because of the lack of commercialization in some cases, and the need for validation in others. Also, many of these new formats are still under development. New research in this eld is now focused on improving robustness, stability and sensitivity by using nanomaterials. Further, biological tools need to be improved in order to obtain multiplexed systems. Finally, the last step in development is yet to be carried out in biosensing (i.e. remote control systems for on-site and on-line analysis). Acknowledgments This study was funded by the Spanish Ministry of Education and Science the project CEMAGUA (CGL200764551/HID). References
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Trends in Green Analytical Chemistry for Aquatic Environment Pollution Analysis

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Trends in Analytical Chemistry, Vol. 29, No.

Trends in Analytical Chemistry, Vol. 29, No. 11, 2010

Trends in Analytical Chemistry, Vol. 29, No. 11, 2010

Trends in Analytical Chemistry, Vol. 29, No. 11, 2010

Trends in Analytical Chemistry, Vol. 29, No. 11, 2010

Trends in Analytical Chemistry, Vol. 29, No. 11, 2010

Figure 1. General scheme of biosensors.

Trends in Analytical Chemistry, Vol. 29, No. 11, 2010

Whole-cell biosensor/Amperometric detection DNA/Amperometric detection DNA/Amperometric detection

1.4 lg/L of fenitrothion and 1.6 lg/L of EPN

[73] [125] [75]

PAT gene Surfactants

DNA/Impedance measurement Bacteria/Amperometric detection

Trends in Analytical Chemistry, Vol. 29, No. 11, 2010

Trends in Analytical Chemistry, Vol. 29, No. 11, 2010

Trends in Analytical Chemistry, Vol. 29, No. 11, 2010

Trends in Analytical Chemistry, Vol. 29, No. 11, 2010

Trends in Analytical Chemistry, Vol. 29, No. 11, 2010

Trends in Analytical Chemistry, Vol. 29, No. 11, 2010

Greening Liquid Chromatography

Biological techniques Immunochemical Enzyme-linked immunosorbent assays (ELISAs) Biosensors

Trends in Analytical Chemistry, Vol. 29, No. 11, 2010

Trends in Analytical Chemistry, Vol. 29, No. 11, 2010

Trends in Analytical Chemistry, Vol. 29, No. 11, 2010

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