**6. Microorganism-based biosensor array**

A microbial biosensor is an analytical device which integrates microorganism(s) with a physical transducer to generate a measurable signal proportional to the concentration of analytes. In recent years, a large number of microbial biosensors have been developed for environmental, food, and biomedical applications[54].

Enzymes are the most widely used biological sensing element in the fabrication of biosensors. Although purified enzymes have very high specificity for their substrates or inhibitors, their application in biosensors construction may be limited by the tedious, timeconsuming and costly enzyme purification, requirement of multiple enzymes to generate the measurable product or need of cofactor/coenzyme. Microorganisms provide an ideal alternative to these bottle-necks. The many enzymes and co-factors that co-exist in the cells give the cells the ability to consume and hence detect large number of chemicals; however, this can compromise the selectivity. They can be easily manipulated and adapted to consume and degrade new substrate under certain cultivating condition. Additionally, the progress in molecular biology/recombinant DNA technologies has opened endless possibilities of tailoring the microorganisms to improve the activity of an existing enzyme or express foreign enzyme/protein in host cell. All of these make microbes excellent biosensing elements[55].

Microorganism-based biosensor arrays classically used for environmental biosensing are mainly bacteria and yeasts, and to a lesser extent algae. Various strains have been exploited, from commercial and well-characterized cells harboring a broad range of substrates to genetically engineered organisms specially constructed to detect specific molecules or groups of molecules, passing through environmental cells isolated from polluted sites offering greater robustness and more specific enzymatic properties[56].

Rapid identification of *Escherichia coli* strains is an important diagnostic goal in applied medicine as well as the environmental and food sciences. Mikkelsen et al.[57] reported an electrochemical, screen-printed biosensor array, where selective recognition is accomplished using lectins that recognize and bind to cell-surface lipopolysaccharides and coulometric transduction exploits non-native external oxidants to monitor respiratory cycle activity in lectin-bound cells. Ten different lectins were separately immobilized onto porous

Biosensor Arrays for Environmental Monitoring 383

based flow cell could be used to design a simple impedance biosensor for the sensitive

Fig. 14. Twenty five spots, 1 nl each, of strain SM118 in ectoine, printed onto the wells of 96 well plate with an APTES coated glass bottom. Reprinted from ref. 58 with permission by

Worldwide herbicide discharge into the aquatic environment is also a growing concern. Adverse effects induced by herbicide contamination are impacting a great variety of organisms and ecosystems, ranging from the primary producers to animals and humans. Biosensors for the rapid detection of herbicides in the environment have also been explored. A multiple-strain algal biosensor was constructed for the detection of herbicides inhibiting photosynthesis by Podola et al.[60]. Nine different microalgal strains were immobilized on an array biochip using permeable membranes. The biosensor allowed on-line measurements of aqueous solutions passing through a flow cell using chlorophyll uorescence as the biosensor response signal. The herbicides atrazine, simazine, diuron, isoproturon and paraquat were detectable within minutes at minimal LOEC (Lowest Observed Effect Concentration) ranging from 0.5 to 100 µg/L, depending on the herbicide and algal strain. The most sensitive strains in terms of EC50 values were Tetraselmis cordiformis and Scherffelia dubia. Less sensitive species were Chlorella vulgaris, Chlamydomonas sp. and Pseudokirchneriella subcapitata, but for most of the strains no general sensitivity or resistance was found. The different responses of algal strains to the five herbicides constituted a complex response pattern (RP), which was analyzed for herbicide specificity

Recombinant bioluminescent bacterial strains are increasingly receiving attention as environmental biosensors due to their advantages, such as high sensitivity and selectivity, low costs, ease of use and short measurement times. Gu et al.[61] use a cell-based array technology that uses recombinant bioluminescent bacteria to detect and classify environmental toxicity followed by developing two biosensor arrays, i.e., a chip and a plate array. Twenty recombinant bioluminescent bacteria, having different promoters fused with the bacterial lux genes, were immobilized within LB-agar. About 2 μl of the cell-agar mixture was deposited into the wells of either a cell chip or a 384-well plate. The bioluminescence (BL) from the cell arrays was measured with the use of highly sensitive cooled CCD camera that measured the bioluminescent signal from the immobilized cells and then quantified the pixel density using image analysis software. The responses from the

detection of bacterial growth and their metabolites.

the Royal Society of Chemistry.

within the linear dose-response relationship.

membranes that feature activated surfaces. Modified membranes were exposed to untreated *E. coli* cultures for 30 min, rinsed, and layered over the individual screen-printed carbon electrodes of the sensor array. The membranes were incubated 5 min in a reagent solution that contained the oxidants menadione and ferricyanide as well as the respiratory substrates succinate and formate. Electrochemical oxidation of ferrocyanide for 2 min provided chronocoulometric data related to the quantities of bound cells. These screen-printed sensor arrays were used in conjunction with factor analysis for the rapid identification of four *E. coli* subspecies (*E. coli* B, *E. coli* Neotype, *E. coli* JM105 and *E. coli* HB101). Systematic examination of lectin-binding patterns showed that these four *E. coli* subspecies are readily distinguished using only five essential lectins.

The last decade has witnessed a significant increase in interest in whole-cell biosensors for diverse applications, as well as a rapid and continuous expansion of array technologies. The combination of these two disciplines has yielded the notion of whole-cell array biosensors. Belkin et al.[58] presented a potential manifestation of this idea by describing the printing of a whole-cell bacterial bioreporters array (Figure 14). Exploiting natural bacterial tendency to adhere to positively charged abiotic surfaces, they describe immobilization and patterning of bacterial ''spots'' in the nanoliter volume range by a non-contact robotic printer. They show that the printed Escherichia coli-based sensor bacteria are immobilized on the surface, and retain their viability and biosensing activity for at least 2 months when kept at 4℃. Immobilization efficiency was improved by manipulating the bacterial genetics, the growth and the printing media and by a chemical modification of the inanimate surface. The result suggests that the methodology presented by them may be applicable to the manufacturing of whole-cell sensor arrays for diverse high throughput applications. In the course of the study, they have also described a novel specific reporter for the detection of respiratory inhibitors. Sodium azide, a chemical with a constantly increasing world distribution, served as the model toxicant. The sensor's response was rapid (20 minutes after exposure) and dose-dependent, and could be maintained for at least 2 months at 4 ℃.

Li et al.[59] developed a double interdigitated array microelectrodes (IAM)-based flow cell for an impedance biosensor to detect viable *Escherichia coli* O157:H7 cells after enrichment in a growth medium. Their study was aimed at the design of a simple flow cell with embedded IAM which does not require complex microfabrication techniques and can be used repeatedly with a simple assembly/disassembly step. The flow cell was also unique in having two IAM chips on both top and bottom surfaces of the flow cell, which enhances the sensitivity of the impedance measurement. *E. coli* O157:H7 cells were grown in a low conductivity yeast–peptone–lactose–TMAO (YPLT) medium outside the flow cell. After bacterial growth, impedance was measured inside the flow cell. Equivalent circuit analysis indicated that the impedance change caused by bacterial growth was due to double layer capacitance and bulk medium resistance. Both parameters were a function of ionic concentration in the medium, which increased during bacterial growth due to the conversion of weakly charged substances present in the medium into highly charged ions. The impedance biosensor successfully detected *E. coli* O157:H7 in a range from 8.0 to 8.2×108 CFU/mL after an enrichment growth of 14.7 and 0.8 h, respectively. A logarithmic linear relationship between detection time (TD) in h and initial cell concentration (N0) in CFU/mL was TD = −1.73 log N0 + 14.62, with R2= 0.93. Double IAM-based flow cell was more sensitive than single IAM-based ow cell in the detection of *E. coli* O157:H7 with 37–61% more impedance change for the frequency range from 10 Hz to 1 MHz. The double IAM-

membranes that feature activated surfaces. Modified membranes were exposed to untreated *E. coli* cultures for 30 min, rinsed, and layered over the individual screen-printed carbon electrodes of the sensor array. The membranes were incubated 5 min in a reagent solution that contained the oxidants menadione and ferricyanide as well as the respiratory substrates succinate and formate. Electrochemical oxidation of ferrocyanide for 2 min provided chronocoulometric data related to the quantities of bound cells. These screen-printed sensor arrays were used in conjunction with factor analysis for the rapid identification of four *E. coli* subspecies (*E. coli* B, *E. coli* Neotype, *E. coli* JM105 and *E. coli* HB101). Systematic examination of lectin-binding patterns showed that these four *E. coli* subspecies are readily

The last decade has witnessed a significant increase in interest in whole-cell biosensors for diverse applications, as well as a rapid and continuous expansion of array technologies. The combination of these two disciplines has yielded the notion of whole-cell array biosensors. Belkin et al.[58] presented a potential manifestation of this idea by describing the printing of a whole-cell bacterial bioreporters array (Figure 14). Exploiting natural bacterial tendency to adhere to positively charged abiotic surfaces, they describe immobilization and patterning of bacterial ''spots'' in the nanoliter volume range by a non-contact robotic printer. They show that the printed Escherichia coli-based sensor bacteria are immobilized on the surface, and retain their viability and biosensing activity for at least 2 months when kept at 4℃. Immobilization efficiency was improved by manipulating the bacterial genetics, the growth and the printing media and by a chemical modification of the inanimate surface. The result suggests that the methodology presented by them may be applicable to the manufacturing of whole-cell sensor arrays for diverse high throughput applications. In the course of the study, they have also described a novel specific reporter for the detection of respiratory inhibitors. Sodium azide, a chemical with a constantly increasing world distribution, served as the model toxicant. The sensor's response was rapid (20 minutes after exposure) and dose-dependent, and could be

Li et al.[59] developed a double interdigitated array microelectrodes (IAM)-based flow cell for an impedance biosensor to detect viable *Escherichia coli* O157:H7 cells after enrichment in a growth medium. Their study was aimed at the design of a simple flow cell with embedded IAM which does not require complex microfabrication techniques and can be used repeatedly with a simple assembly/disassembly step. The flow cell was also unique in having two IAM chips on both top and bottom surfaces of the flow cell, which enhances the sensitivity of the impedance measurement. *E. coli* O157:H7 cells were grown in a low conductivity yeast–peptone–lactose–TMAO (YPLT) medium outside the flow cell. After bacterial growth, impedance was measured inside the flow cell. Equivalent circuit analysis indicated that the impedance change caused by bacterial growth was due to double layer capacitance and bulk medium resistance. Both parameters were a function of ionic concentration in the medium, which increased during bacterial growth due to the conversion of weakly charged substances present in the medium into highly charged ions. The impedance biosensor successfully detected *E. coli* O157:H7 in a range from 8.0 to 8.2×108 CFU/mL after an enrichment growth of 14.7 and 0.8 h, respectively. A logarithmic linear relationship between detection time (TD) in h and initial cell concentration (N0) in CFU/mL was TD = −1.73 log N0 + 14.62, with R2= 0.93. Double IAM-based flow cell was more sensitive than single IAM-based ow cell in the detection of *E. coli* O157:H7 with 37–61% more impedance change for the frequency range from 10 Hz to 1 MHz. The double IAM-

distinguished using only five essential lectins.

maintained for at least 2 months at 4 ℃.

based flow cell could be used to design a simple impedance biosensor for the sensitive detection of bacterial growth and their metabolites.

Fig. 14. Twenty five spots, 1 nl each, of strain SM118 in ectoine, printed onto the wells of 96 well plate with an APTES coated glass bottom. Reprinted from ref. 58 with permission by the Royal Society of Chemistry.

Worldwide herbicide discharge into the aquatic environment is also a growing concern. Adverse effects induced by herbicide contamination are impacting a great variety of organisms and ecosystems, ranging from the primary producers to animals and humans. Biosensors for the rapid detection of herbicides in the environment have also been explored. A multiple-strain algal biosensor was constructed for the detection of herbicides inhibiting photosynthesis by Podola et al.[60]. Nine different microalgal strains were immobilized on an array biochip using permeable membranes. The biosensor allowed on-line measurements of aqueous solutions passing through a flow cell using chlorophyll uorescence as the biosensor response signal. The herbicides atrazine, simazine, diuron, isoproturon and paraquat were detectable within minutes at minimal LOEC (Lowest Observed Effect Concentration) ranging from 0.5 to 100 µg/L, depending on the herbicide and algal strain. The most sensitive strains in terms of EC50 values were Tetraselmis cordiformis and Scherffelia dubia. Less sensitive species were Chlorella vulgaris, Chlamydomonas sp. and Pseudokirchneriella subcapitata, but for most of the strains no general sensitivity or resistance was found. The different responses of algal strains to the five herbicides constituted a complex response pattern (RP), which was analyzed for herbicide specificity within the linear dose-response relationship.

Recombinant bioluminescent bacterial strains are increasingly receiving attention as environmental biosensors due to their advantages, such as high sensitivity and selectivity, low costs, ease of use and short measurement times. Gu et al.[61] use a cell-based array technology that uses recombinant bioluminescent bacteria to detect and classify environmental toxicity followed by developing two biosensor arrays, i.e., a chip and a plate array. Twenty recombinant bioluminescent bacteria, having different promoters fused with the bacterial lux genes, were immobilized within LB-agar. About 2 μl of the cell-agar mixture was deposited into the wells of either a cell chip or a 384-well plate. The bioluminescence (BL) from the cell arrays was measured with the use of highly sensitive cooled CCD camera that measured the bioluminescent signal from the immobilized cells and then quantified the pixel density using image analysis software. The responses from the

Biosensor Arrays for Environmental Monitoring 385

biosensor array can be easily adapted for continuous monitoring operations by integration with a computer-controlled sampler to format automatic analytical system. Because of these advantages, more and more biosensor arrays are applied in varied areas including environmental monitoring. An overview of the applications for environment by using

Target Biosensor array type LOD Reference

10-6 mol/L [65]

CFU/mL [68]

107 CFU/mL [72]

CFU/mL [75]

Subclasses Array of photosystem II mutants 3×10-9 mol/L [64]

array

agents Planar waveguide biosensor array 5×105

array

*Shigella dysenteriae* Antibody-based biosensor array 5×104

Table 1. Applications of biosensor arrays for environmental monitoring

[1] G. Hanrahan, D.G. Patil, J. Wang, J Environ Monitor, 6 (2004) 657-664. [2] M. Badihi-Mossberg, V. Buchner, J. Rishpon, Electroanal, 19 (2007) 2015-2028. [3] A. Cavalcanti, B. Shirinzadeh, M. Zhang, L. Kretly, Sensors, 8 (2008) 2932-2958.

F.S. Ligler, Sensor Actuat B-Chem, 113 (2006) 599-607.

C.J. Myatt, Anal Sci, 23 (2007) 5-10.

*Yersinia pestis* F1 Antibody-based biosensor array 25 ng/mL [73] *Bacillus globigii* Antibody-based biosensor array 105 CFU/mL [74]

Despite the high number of biosensor arrays under development and the amount of research literature on this area, few practical systems are currently enjoying market acceptance for environmental applications. The Naval Research Laboratory (NRL) biosensor arrays are the most successful type of biosensor arrays that have found commercial application not only in environmental monitoring but also in the monitoring of biomolecular interaction events in general. Biosensor arrays still need more research and development in order to achieve the stability, sensitivity, specificity, and versatility that will attract confidence of potential users, especially for biotechnology and environmental

[4] K.E. Sapsford, M.M. Ngundi, M.H. Moore, M.E. Lassman, L.C. Shriver-Lake, C.R. Taitt,

[5] M. Seidel, R. Niessner, Analytical and Bioanalytical Chemistry, 391 (2008) 1521-1544. [6] F.S. Ligler, K.E. Sapsford, J.P. Golden, L.C. Shriver-Lake, C.R. Taitt, M.A. Dyer, S. Barone,

Microbial species Electrochemical biosensor array Not given [66] *Escherichia coli* Quantum dot-based array 10 CFU/mL [67]

aflatoxin B1 NRL biosensor array 0.6 ng/g [69] Ochratoxin A Antibody-based biosensor array 3.8 ng/g [70] Odour Colorimetric biosensor array Not given [71]

biosensor arrays, which are not mentioned in this review, are listed in Table 1.

Metal ions All-solid-state potentiometric biosensor

*Escherichia coli* Antimicrobial Peptides based biosensor

Herbicide

Bio-hazardous

applications.

**8. References** 

cell arrays were characterized using three chemicals that cause either superoxide damage (paraquat), DNA damage (mitomycin C) or protein/membrane damage (salicylic acid). The responses were found to be dependent upon the promoter fused upstream of the lux operon within each strain. Therefore, a sample's toxicity can be analyzed and classified through the changes in the BL expression from each well. Moreover, a time of only 2 h was needed for analysis, making either of these arrays a fast, portable and economical high-throughput biosensor system for detecting environmental toxicities.

Because of their ability to perform functional sensing, living cell-based biosensors are drawing increased attention. The work reported by Walt et al.[62] demonstrates the ability to fabricate an optical imaging fiber-based living bacterial cell array for genotoxin detection. A biosensor composed of a high-density living bacterial cell array was fabricated by inserting bacteria into a micro-well array formed on one end of an imaging fiber bundle. The size of each micro-well allows only one cell to occupy each well. In this biosensor, *E. coli* cells carrying a *recA::gfp* fusion were used as sensing components for genotoxin detection. Each fiber in the array has its own light pathway, enabling thousands of individual cell responses to be monitored simultaneously with both spatial and temporal resolution. The biosensor was capable of performing cell-based functional sensing of a genotoxin with high sensitivity and short incubation times (1 ng/mL mitomycin C after 90 min). The biosensors demonstrated an active sensing lifetime of more than 6 h and a shelf lifetime of two weeks. Their group reported another live cell biosensor array[63], which was fabricated by immobilizing bacterial cells on the face of an optical imaging fiber containing a high density array of micro-wells. Each microwell accommodates a single bacterium that was genetically engineered to respond to a specic analyte. A genetically modified *Escherichia coli* strain, containing the *lacZ* reporter gene fused to the heavy metal-responsive gene promoter *zntA*, was used to fabricate a mercury biosensor. A plasmid carrying the gene coding for the enhanced cyan fluorescent protein (ECFP) was also introduced into this sensing strain to identify the cell locations in the array. Single cell *lacZ* expression was measured when the array was exposed to mercury and a response to 100 nmol/L Hg2+ could be detected after a 1-h incubation time. The optical imaging fiber-based single bacterial cell array is a flexible and sensitive biosensor platform that can be used to monitor the expression of different reporter genes and accommodate a variety of sensing strains.

#### **7. Conclusion and future direction**

In recent years, there have been dramatic advances in a new analytical format, the biosensor array, a tool that has revolutionized our ability to characterize and quantify biologically and envitonmetally relevant molecules. The biosensor arrays address the need for rapid, sensitive, and specific screening for multiple pollutants at the site of sample collection. The biosensor arrays have several very significant advantages for such applications: (1) The number of analyte which can be detected simultaneously can be expanded as need dictates and specific analyte become available. (2) The biosensor arrays and tracer reagents are reusable if no target agent binds to the array surface. This feature significantly decreases the cost and operational burden for the user and simplifies automation for extended monitoring applications. (3) The biosensor array is simple to use. It is easily portable for first responder applications. The insertion of the sensor array, tracer reagents and samples is very simple with no requirement for alignment operations by the user. (4) The biosensor array is a lowcost system which can be made even more cost effective with mass production. (5) The

cell arrays were characterized using three chemicals that cause either superoxide damage (paraquat), DNA damage (mitomycin C) or protein/membrane damage (salicylic acid). The responses were found to be dependent upon the promoter fused upstream of the lux operon within each strain. Therefore, a sample's toxicity can be analyzed and classified through the changes in the BL expression from each well. Moreover, a time of only 2 h was needed for analysis, making either of these arrays a fast, portable and economical high-throughput

Because of their ability to perform functional sensing, living cell-based biosensors are drawing increased attention. The work reported by Walt et al.[62] demonstrates the ability to fabricate an optical imaging fiber-based living bacterial cell array for genotoxin detection. A biosensor composed of a high-density living bacterial cell array was fabricated by inserting bacteria into a micro-well array formed on one end of an imaging fiber bundle. The size of each micro-well allows only one cell to occupy each well. In this biosensor, *E. coli* cells carrying a *recA::gfp* fusion were used as sensing components for genotoxin detection. Each fiber in the array has its own light pathway, enabling thousands of individual cell responses to be monitored simultaneously with both spatial and temporal resolution. The biosensor was capable of performing cell-based functional sensing of a genotoxin with high sensitivity and short incubation times (1 ng/mL mitomycin C after 90 min). The biosensors demonstrated an active sensing lifetime of more than 6 h and a shelf lifetime of two weeks. Their group reported another live cell biosensor array[63], which was fabricated by immobilizing bacterial cells on the face of an optical imaging fiber containing a high density array of micro-wells. Each microwell accommodates a single bacterium that was genetically engineered to respond to a specic analyte. A genetically modified *Escherichia coli* strain, containing the *lacZ* reporter gene fused to the heavy metal-responsive gene promoter *zntA*, was used to fabricate a mercury biosensor. A plasmid carrying the gene coding for the enhanced cyan fluorescent protein (ECFP) was also introduced into this sensing strain to identify the cell locations in the array. Single cell *lacZ* expression was measured when the array was exposed to mercury and a response to 100 nmol/L Hg2+ could be detected after a 1-h incubation time. The optical imaging fiber-based single bacterial cell array is a flexible and sensitive biosensor platform that can be used to monitor the expression of different

In recent years, there have been dramatic advances in a new analytical format, the biosensor array, a tool that has revolutionized our ability to characterize and quantify biologically and envitonmetally relevant molecules. The biosensor arrays address the need for rapid, sensitive, and specific screening for multiple pollutants at the site of sample collection. The biosensor arrays have several very significant advantages for such applications: (1) The number of analyte which can be detected simultaneously can be expanded as need dictates and specific analyte become available. (2) The biosensor arrays and tracer reagents are reusable if no target agent binds to the array surface. This feature significantly decreases the cost and operational burden for the user and simplifies automation for extended monitoring applications. (3) The biosensor array is simple to use. It is easily portable for first responder applications. The insertion of the sensor array, tracer reagents and samples is very simple with no requirement for alignment operations by the user. (4) The biosensor array is a lowcost system which can be made even more cost effective with mass production. (5) The

biosensor system for detecting environmental toxicities.

reporter genes and accommodate a variety of sensing strains.

**7. Conclusion and future direction** 

biosensor array can be easily adapted for continuous monitoring operations by integration with a computer-controlled sampler to format automatic analytical system. Because of these advantages, more and more biosensor arrays are applied in varied areas including environmental monitoring. An overview of the applications for environment by using biosensor arrays, which are not mentioned in this review, are listed in Table 1.


Table 1. Applications of biosensor arrays for environmental monitoring

Despite the high number of biosensor arrays under development and the amount of research literature on this area, few practical systems are currently enjoying market acceptance for environmental applications. The Naval Research Laboratory (NRL) biosensor arrays are the most successful type of biosensor arrays that have found commercial application not only in environmental monitoring but also in the monitoring of biomolecular interaction events in general. Biosensor arrays still need more research and development in order to achieve the stability, sensitivity, specificity, and versatility that will attract confidence of potential users, especially for biotechnology and environmental applications.
