 |
 |
 |
 |
 |
 |
|
Sains Malaysiana 54(5)(2025): 1375-7392
http://doi.org/10.17576/jsm-2025-5405-14
Biosensor Berasaskan Sel E. coli Terubahsuai Protein Pendarfluor
Hijau (GFP E. coli) Terpegun Mikrosfera Alginat untuk Pengukuran Ketoksikan Air
(Biosensor Based
on Green Fluorescent Protein-Modified E. coli(GFP E. coli) Cells-Immobilised Alginate Microspheres for Water Toxicity
Measurements)
DEDI FUTRA1,2, LING LING TAN3,
SALMIJAH SURIF1 & LEE YOOK HENG1,*
1Jabatan Sains Kimia, Fakulti Sains dan Teknologi, Universiti Kebangsaan Malaysia,
43600 UKM Bangi, Selangor, Malaysia
2Department
of Chemistry Education, Faculty of Education, Universitas Riau,
Kampus Binawidya KM 12.5 Pekanbaru, 28131,
Riau, Indonesia
3Pusat
Kajian Bencana Asia Tenggara (SEADPRI), Institut Alam Sekitar dan
Pembangunan (LESTARI), Universiti Kebangsaan Malaysia,
43600 UKM Bangi, Selangor, Malaysia
Received: 25 October 2024/Accepted:
29 December 2024
Abstrak
Biosensor ketoksikan optik telah direka bentuk berasaskan mikroorganisma
sel keseluruhan terpegun iaitu strain DH5α Escherichia coli terubah
suai protein pendarfluor hijau (GFP E. coli) dalam mikrosfera alginat
melalui mikroenkapsulasi untuk pengesanan bahan toksik, seperti logam berat
[Cu(II), Cd(II), Pb(II), Zn(II), Cr(VI), Co(II), Ni(II), Ag(I) dan Fe(III)], racun
rumpai [2,4-asid diklorofenoksiasetik (2,4-D)] dan racun serangga (klorpirifos).
Biosensor bakteria yang teguh dan mudah alih telah dicirikan dengan kaedah
spektrofluorimetrik gentian optik. Kehadiran bahan pencemar alam sekitar telah
mengubah ciri aruhan pendarfluor GFP E. coli dan mengakibatkan perubahan
fotokimia. Rangsangan biosensor diukur pada panjang gelombang pengujaan dan
pemancaran pendarfluor optimum masing-masing pada 400±2 nm dan 485±2 nm. Had
pengesanan (LOD) biosensor untuk logam berat, racun rumpai dan racun serangga
diperoleh masing-masing pada 0.06-2900.00 μg/L, 0.07 μg/L dan 36.50
μg/L. Ujian kebolehulangan biosensor menunjukkan sisihan piawai relatif
(RSD) purata pada <5.0% dan rangsangan biosensor adalah stabil sehingga enam
minggu. Biosensor memberikan ransangan perencatan terhadap toksik tunggal dalam
susunan Cu(II)>klorpirifos>Cd(II)>Pb(II)>Zn(II)>2,4-D>Cr(VI)>Co(II)>Ni(II)>
Ag(I)>Fe(III). Biosensor mikrob yang dibangunkan menunjukkan rangsangan
antagonis untuk campuran ketoksikan dan ia boleh mengukur kehadiran bahan
toksik campuran ini pada tahap antagonis 86-100%. Biosensor gentian optik
berasaskan mikrosfera alginat telah dicirikan untuk pengesanan tahap pencemaran
toksik dalam sampel air persekitaran. Keputusan penentusahan yang diperoleh
menggunakan mikrosfera alginat terubah suai GFP E. coli adalah setanding
dengan keputusan yang diperoleh menggunakan kaedah spektroanalisis seperti
spektroskopi serapan atom (AAS) dan kromatografi gas-spektroskopi jisim
(GC-MS).
Kata kunci: biosensor gentian optik; biosensor sel keseluruhan; GFP E.
coli; logam berat; mikrosfera alginat
Abstract
An optical toxicity biosensor has been designed based on immobilised whole-cell microorganisms i.e., green
fluorescent protein-modified DH5α strain of Escherichia coli (GFP E.
coli) in the alginate microspheres via microencapsulation for detection of
toxicants, such as heavy metals [Cu(II), Cd(II), Pb(II), Zn(II), Cr(VI),
Co(II), Ni(II), Ag(I), and Fe(III)], herbicide [2,4-dichlorophenoxyacetic acid
(2,4-D)], and insecticide (chlorpyrifos). The robust and mobile bacterial
biosensor was characterised with the fiber optic
spectrofluorimetric method. The presence of environmental pollutants altered
the GFP E. coli fluorescence induction characteristic and resulted in
photochemical changes. The biosensor response was measured at optimal
fluorescence excitation and emission wavelengths at 400±2 nm and 485±2 nm,
respectively. The limit of detections
(LOD) of the biosensor for heavy metals, herbicide, and insecticide were found
to be 0.06-2900 μg/L, 0.07 μg/L,
and 36.5 μg/L, respectively. The reproducibility
test of the biosensor showed an average relative standard deviation (RSD) of
<5.0% and the biosensor response was stable for up to six weeks. The
biosensor gave inhibition response toward single toxicant in the order of
Cu(II)>chlorpyrifos>Cd(II)>Pb(II)>Zn(II)>2,4-D>Cr(VI)>Co(II)>Ni(II)
>Ag(I)>Fe(III). The developed microbial biosensor showed antagonistic
results for toxicity mixture, whereby it can quantify the presence of these
mixed toxicants at 86–100% antagonistic level. The alginate microspheres-based
optical fiber biosensor has been characterised for
the detection of toxicant contamination levels in environmental water samples.
The validation results obtained using GFP E. coli-modified alginate
microspheres were in good agreement with those obtained using spectroanalytical methods e.g., atomic absorbance
spectroscopy (AAS) and gas chromatography-mass spectroscopy (GC-MS).
Keywords: Alginate microspheres; GFP E.
coli; heavy metal; optical fiber biosensor; whole cell biosensor
REFERENCES
Abasalizadeh, F., Moghaddam, S.V., Alizadeh, E., Akbari, E., Kashani, E., Fazljou,
S.M.B., Torbati, M. & Akbarzadeh, A. 2020. Alginate-based hydrogels as drug delivery vehicles in cancer treatment
and their applications in wound dressing and 3D bioprinting. Journal of
Biological Engineering 14: 8.
https://doi.org/10.1186/s13036-020-0227-7
Ahmed, S.A. 2008. Invertase production
by Bacillus macerans immobilized on calcium
alginate beads. Journal of Applied Sciences 4(12): 1777-1781.
Arias-Barreiro, C.R., Okazaki, K., Koutsaftis, A., Inayat-Hussain, S.H., Tani, A., Katsuhara, M., Kimbara, K. &
Mori, I.C. 2010. A bacterial biosensor for oxidative stress using the
constitutively expressed redox-sensitive protein roGFP2. Sensors 10(7):
6290-6306. doi: 10.3390/s100706290
Broerse, M. & Gestel, C.A.M.V. 2020. Mixture effects of nickel and chlorpyrifos on Folsomia candida (Collembola) explained from
development of toxicity in time. Chemosphere 79(9): 953-957. doi: 10.1016/j.chemosphere.2010.02.032
Brown, J.Q., Srivastava, R. &
McShane, M.J. 2005. Encapsulation of glucose oxidase and an oxygen-quenched
fluorophore in poly-electrolyte-coated calcium alginate microspheres as optical
glucose sensor systems. Biosensors and Bioelectronics 21(1): 212-216. https://doi.org/10.1016/j.bios.2004.08.020
Cano-Vicent, A., Tuñón-Molina, A., Bakshi, H., Serra, R.S.I., Alfagih, I.M., Tambuwala, M.M.
& Serrano-Aroca, A. 2023. Biocompatible alginate film crosslinked with Ca2+ and Zn2+ possesses antibacterial, antiviral, and anticancer
activities. ACS Omega 8(27): 24396-24405.
https://doi.org/10.1021/acsomega.3c01935
Chakraborty, T., Babu, P.G., Alam, A. &
Chaudhari, A. 2008. GFP expressing bacterial biosensor to measure lead
contamination in aquatic environment. Current Science 94(6): 800-805.
Chan, L.W. Jin,
Y. & Heng, P.W.S. 2002. Cross-linking mechanisms of calcium and zinc in
production of alginate microspheres. International Journal of Pharmaceutics 242(1-2): 255-258. https://doi.org/10.1016/S0378-5173(02)00169-2
Chen, S., Chen, X., Su, H.,
Guo, M. & Liu, H. 2023. Advances in synthetic-biology-based whole-cell
biosensors: Principles, genetic modules, and applications in food safety. International Journal of Molecular
Sciences 24(9):
7989. doi: 10.3390/ijms24097989
Cui, Z., Luan, X., Jiang, H., Li,
Q., Xu, G., Sun, C., Song, Y., Davison, P.A. & Huang, W.E. 2018.
Application of a bacterial whole cell biosensor for the rapid detection of
cytotoxicity in heavy metal contaminated seawater. Chemosphere 200:
322-329. doi: 10.1016/j.chemosphere.2018.02.097
Dawson, J.J.C., Campbell, C.D.,
Towers, E., Cameron, C.M. & Paton, G.I. 2006. Linking biosensor responses
to Cd, Cu, and Zn partitioning in soils. Environmental Pollution 142(3):
493-500. https://doi.org/10.1016/j.envpol.2005.10.029
Deng, X., Xia, Y., Hu, W., Zhang,
H. & Shen, Z. 2010. Cadmium-induced oxidative damage and protective effects
of N-acetyl-L-cysteine against cadmium toxicity in Solanum nigrum L. Journal
of Hazardous Materials 180(1-3): 722-729. doi:
10.1016/j.jhazmat.2010.04.099
Echeverri-Jaramillo, G.,
Jaramillo-Colorado, B., Sabater-Marco, C. & Castillo-López, M.A. 2020.
Acute toxicity of chlorpyrifos and its metabolite 3, 5, 6-trichloro-2-pyridinol
alone and in combination using a battery of bioassays. Environmental Science
and Pollution Research 27(26): 32770-32778. doi: 10.1007/s11356-020-09392-x
Futra, D., Lee, Y.H., Surif, S., Musa, A. & Tan, L.L. 2014. Microencapsulated Aliivibrio fischeri in alginate microspheres for monitoring heavy metal toxicity in environmental
waters. Sensors 14(12): 23248-23268. https://doi.org/10.3390/s141223248
Guler, M., Turkoglu, V. & Basi,
Z. 2017. Determination of malation, methidathion, and
chlorpyrifos ethyl pesticides using acetyl-cholinesterase biosensor based on Nafion/Ag@ rGO-NH2 nanocomposites. Electrochimica Acta 240: 129-135.
https://doi.org/10.1016/j.electacta.2017.04.069
Hanson, G.T., Aggeler, R.,
Oglesbee, D., Cannon, M., Capaldi, R.A., Tsien, R.Y. & Remington, S.J.
2004. Investigating mitochondrial redox potential with redox-sensitive green
fluorescent protein indicators. Journal of Biological Chemistry 279(13):
13044-13053. doi: 10.1074/jbc.M312846200
He, J., Zhang, X., Qian, Y., Wang,
Q. & Bai, Y. 2022. An engineered quorum-sensing-based whole-cell biosensor
for active degradation of organophosphates. Biosensors and Bioelectronics 206: 114085. doi: 10.1016/j.bios.2022.114085
Hu, N. & Sun, H. 2019. A
biosensor for hydrogen peroxide based on myoglobin immobilized on calcium
alginate microspheres. Material Sciences 9(2): 170-176. doi:10.12677/MS.2019.92022
Jain, M., Yadav, P., Joshi, B.,
Joshi, A. & Kodgire, P. 2021. Recombinant
organophosphorus hydrolase (OPH) expression in E. coli for the effective
detection of organophosphate pesticides. Protein Expression and Purification 186: 105929. doi: 10.1016/j.pep.2021.105929
Katsanakis, N., Katsivelis,
A. & Kintzois, S. 2009. Immobilization of electroporated for fabrication
cellular biosensors; physiological effect of the shape of calcium alginate
matrices and foetal calf serum. Sensors 9(1):
378-385. doi: 10.3390/s90100378
Khattab, T.A., Fouda, M.M., Abdelrahman,
M.S., Othman, S.L., Bin-Jumah, M., Alqaraawi, M.A., Fassam, H.A. & Allam, A.A. 2019. Co-encapsulation of
enzyme and tricyanofuran hydrazone into alginate microcapsules incorporated onto cotton fabric as a biosensor for
colorimetric recognition of urea. Reactive and Functional Polymers 142:
199-206. https://doi.org/10.1016/j.reactfunctpolym.2019.06.016
Khatun, M.A., Hoque, M.A., Zhang,
Y., Lu, T., Cui, L., Zhou, N.Y. & Feng, Y. 2018. Bacterial consortium-based
sensing system for detecting organophosphorus pesticides. Analytical
Chemistry 90(17): 10577-10584. doi:
10.1021/acs.analchem.8b02709
Kim, B.C. & Gu, M.B. 2003. A
bioluminescent sensor for high throughput toxicity classification. Biosensors and Bioelectronics 18(8):
1015-1021. doi: 10.1016/s0956-5663(02)00220-8
Kumar, A., Kothari, A., Kumar, P.,
Singh, A., Tripathi, K., Gairolla, J., Pai, M. &
Omar, B.J. 2023. Introduction
to Alginate: Biocompatible, Biodegradable, Antimicrobial Nature and Various
Applications. United Kindom: IntechOpen Limited. doi: 10.5772/intechopen.110650
Lindahl, P.A. 2012. Metal–metal
bonds in biology. Journal of Inorganic Biochemistry 106(1): 172-178. doi: 10.1016/j.jinorgbio.2011.08.012
Martinez, A.R., Heil, J.R. &
Charles, T.C. 2019. An engineered GFP fluorescent bacterial biosensor for
detecting and quantifying silver and copper ions. Biometals 32(2): 265-272. doi: 10.1007/s10534-019-00179-3
Mohseni, M., Abbaszadeh, J., Maghool, S.S. & Chaichi, M.J. 2018. Heavy metals detection using
biosensor cells of a novel marine luminescent bacterium Vibrio sp. MM1
isolated from the Caspian Sea. Ecotoxicology and Environmental Safety 148: 555-560. https://doi.org/10.1016/j.ecoenv.2017.11.002
Momplaisir, G., Rosal, C.G., Heithmar, E.M.,
Varner, K.E., Riddick, L.A., Bradford, D.F. & Tallent-Halsell,
N.G. 2010. Development of a solid phase extraction method for agricultural
pesticides in large-volume water samples. Talanta 81(4-5): 1380-1386. doi:
10.1016/j.talanta.2010.02.038
Moraskie, M., Roshid,
M.H.O., O’Connor, G., Dikici, E., Zingg, J., Deo, S. & Daunert,
S. 2021. Microbial whole-cell biosensors: Current applications, challenges, and
future perspectives. Biosensors and Bioelectronics 191: 113359.
https://doi.org/10.1016/j.bios.2021.113359
Nourmohammadi, E., Hosseinkhani,
S., Nedaeinia, R., Khoshdel-Sarkarizi,
H., Nedaeinia, M., Ranjbar, M., Ebrahimi, N., Farjami, Z., Nourmohammdi, M.,
Mahmoudi, A., Goli, M., Ferns, G.A. & Sadeghizadeh,
M. 2020. Construction of a sensitive and specific lead biosensor using a
genetically engineered bacterial system with a luciferase gene reporter
controlled by pbr and cadA promoters. Biomedical Engineering Online 19(1): 79. doi:
10.1186/s12938-020-00816-w
Ooi, L., Lee, Y.H. & Mori,
I.C. 2015. A high-throughput oxidative stress biosensor based on Escherichia
coli roGFP2 cells immobilized in a k-carrageenan matrix. Sensors 15(2): 2354-2368. doi: 10.3390/s150202354
Phoong, S.Y. & Phoong, S.W. 2021. Inferential Statistics: Hypothesis
Testing, Quantitative Data Analysis Using SPSS Statistic. Malaysia:
Malaysian Scholarly Publishing Council.
Prasad, J., Joshi, A., Jayant,
R.D. & Srivastava, R. 2011. Cholesterol biosensors based on oxygen sensing
alginate-silica microspheres. Biotechnology and Bioengineering 108: 2011-2021. doi:
10.1002/bit.23143
Rathnayake, I.V.N., Megharaj,
M. & Naidu, R. 2021. Green fluorescent protein based whole cell
bacterial biosensor for the detection
of bioavailable heavy metals in soil environment. Environmental Technology
& Innovation 23: 101785. https://doi.org/10.1016/j.eti.2021.101785
Raus, R.A., Nawawi, F.M.F.W. & Nasaruddin, R.R. 2021. Alginate and alginate composites for
biomedical applications. Asian Journal of Pharmaceutical Sciences 16(3):
280-306. https://doi.org/10.1016/j.ajps.2020.10.001
Ritcharoon, B., Sallabhan,
R., Toewiwat, N., Mongkolsuk,
S. & Loprasert, S. 2020. Detection of
2,4-dichlorophenoxyacetic acid herbicide using a FGE-sulfatase based whole-cell Agrobacterium biosensor. Journal of Microbiological Methods 175:
105997. doi: 10.1016/j.mimet.2020.105997
Rosochacki, S.J. & Matejczyk,
M. 2002. Green fluorescent protein as a molecular marker in microbiology. Acta Microbiologica Polonica 51(3): 205-216.
Sahoo, D.R.
& Biswal, T. 2021. Alginate
and its application to tissue engineering. SN Applied Sciences 3: 30. https://doi.org/10.1007/s42452-020-04096-w
Schmitz, R.P.H., Eisentrager,
A. & Dott, W. 1999. Agonistic
and antagonistic toxic effect observed with miniaturized growth and
luminescence inhibition assays. Chemosphere 38(1): 79-95. doi: 10.1016/s0045-6535(98)00175-1
Serrano, N., Sestakova,
I., Dıaz-Cruz, J.M. & Arino, C. 2006.
Adsorptive accumulation in constant current stripping chronopotentiometry as an
alternative for the electrochemical study of metal complexation by
thiol-containing peptides. Journal of Electroanalytical Chemistry 591(1):
105-117. https://doi.org/10.1016/j.jelechem.2006.03.038
Silva, C.M., Ribeiro, A.J.,
Figueiredo, I.V., Goncalves, A.R. & Veiga, F. 2006. Alginate microspheres
prepared by internal gelation: Development and effect on insulin stability. International
Journal of Pharmaceutics 311(1-2): 1-10. doi: 10.1016/j.ijpharm.2005.10.050
Silva, L.C., Moreira, R.A., Pinto,
T.J., Ogura, A.P., Yoshii, M.P., Lopes, L.F., Montagner, C.C., Goulart, B.V.,
Daam, M.A. & Espindola, E.I. 2020. Acute and chronic toxicity of 2, 4-D and
fipronil formulations (individually and in mixture) to the Neotropical cladoceran Ceriodaphnia silvestrii. Ecotoxicology 29(9):
1462-1475. doi: 10.1007/s10646-020-02275-4
Sorensen,
S.J., Burmolle, M. & Hansen, L.H. 2006. Making bio-sense of toxicity: New developments in
whole-cell biosensors. Current Opinion in Biotechnology 17(1): 11-16. doi:10.1016/j.copbio.2005.12.007
Sumner, J., Westberg, N.M.,
Stoddard, A.K., Hurst, T.K., Cremer, M., Thompson, R.B., Fierke, C.A. & Kopelman,
R. 2006. DsRed a highly sensitive, selective and
reversible fluorescence-based biosensor for both Cu+ and Cu2+ ions. Biosensors and Bioelectronics 21(7): 1302-1308. doi: 10.1016/j.bios.2005.04.023
Valenzuela-García, L.I., Alarcón-Herrera, M.T., Ayala-García, V.M., Barraza-Salas, M., Salas-Pacheco, J.M., Díaz-Valles, J.F. & Pedraza-Reyes, M. 2023. Environmental Microbiology 11(4): e00432-23. doi:
https://doi.org/10.1128/spectrum.00432-23
Vopálenská, I., Váchová, L. & Palková, Z. 2015. New biosensor for detection of copper
ions in water based on immobilized genetically modified yeast cells. Biosensors
and Bioelectronics 72: 160-167. doi: 10.1016/j.bios.2015.05.006
Wang, G.H., Cheng, C.Y., Tsai,
T.H., Chiang, P.K. & Chung, Y.C. 2021. Highly sensitive luminescent
bioassay using recombinant Escherichia coli biosensor for rapid
detection of low Cr (VI) concentration in environmental water. Biosensors 11(10): 357. https://doi.org/10.3390/bios11100357
Whangsuk, W., Thiengmag,
S., Dubbs, J., Mongkolsuk, S. & Loprasert, S. 2016. Specific detection of the pesticide
chlorpyrifos by a sensitive genetic-based whole cell biosensor. Analytical
Biochemistry 493: 11-13. https://doi.org/10.1016/j.ab.2015.09.022
Wu, Y., Wang, C., Wang, D.
& Wei, N. 2021. A whole-cell biosensor for point-of-care detection of
waterborne bacterial pathogens. ACS Synthetic Biology 10(2): 333-344. https://doi.org/10.1021/acssynbio.0c00491
Yu, D., Bai, L., Zhai, J.,
Wang, Y. & Dong, S. 2017. Toxicity
detection in water containing heavy metal ions with a self-powered microbial
fuel cell-based biosensor. Talanta 168:
210-216. https://doi.org/10.1016/j.talanta.2017.03.048
Yu, D., Zhang,
H., Bai, L., Fang, Y., Liu, C., Zhang, H., Li, T., Han, L., Yu, Y., Hongwen, Y.
& Dong, S. 2020. Visual detection of the toxicity
of wastewater containing heavy metal ions using a microbial fuel cell biosensor
with a Prussian blue cathode. Sensors and Actuators B: Chemical 302:
127177. https://doi.org/10.1016/j.snb.2019.127177
Zevallos-Aliaga,
D., Graeve, S.D., Obando-Chavez, P., Vaccari, N.A., Gao, Y., Peeters, T. &
Guerra, D.G. 2024. Highly
sensitive whole-cell mercury biosensors for environmental monitoring. Biosensors 14(5): 246. https://doi.org/10.3390/bios14050246
*Corresponding author; email: leeyookheng@yahoo.co.uk
|
|||
|
