Nanobiosensors for Pathogenic Agents Detection

Beltrán-Pineda, Mayra; Peña-Solórzano, Diana; Sierra, César Augusto

doi:10.21577/0103-5053.20210081

Mayra Beltrán-Pineda

Doctorado en Biotecnología, Facultad de Ciencias, Universidad Nacional de Colombia, Sede Bogotá, Carrera 45 26-85, A.A 5997, Bogotá, Colombia

Grupo de Investigación en Macromoléculas, Departamento de Química, Universidad Nacional de Colombia, Sede Bogotá, Carrera 45 26-85, A.A 5997, Bogotá, Colombia

Mayra Beltrán-Pineda was born in Tunja-Boyacá, graduated as a Biologist from the Universidad Pedagógica y Tecnológica de Colombia in 2004. She was a young researcher at COLCIENCIAS in 2006. She obtained her Master’s degree in Sciences-Microbiology in 2009 from the Universidad Nacional de Colombia. Her research area is environmental microbiology, biotechnology, and nanotechnology. She is currently a PhD student in Biotechnology from the Universidad Nacional de Colombia under the supervision of Professor Cesar Sierra in the area of nanobiotechnology. Her doctoral research is focused on the biosynthesis of silver nanoparticles for the control of a potato plant pathogen.

Diana Peña-Solórzano

Grupo de Investigación en Macromoléculas, Departamento de Química, Universidad Nacional de Colombia, Sede Bogotá, Carrera 45 26-85, A.A 5997, Bogotá, Colombia

Diana C. Peña-Solórzano was born in Calarcá, Quindío, and graduated in Chemistry from the Universidad Nacional of Colombia (2011). She obtained a double doctoral degree from the Universidad Nacional of Colombia and Regensburg University in Germany (2017). She held a postdoctoral degree at Universidad Nacional of Colombia (2019). Her research interests are in organic chemistry with applications in medicine, including the synthesis, characterization, and inhibitory activity evaluation of new analogs of tariquidar on breast cancer resistance protein (ABCG2). She is also interested in the development of eutectic solvents based on zinc chloride for green chemistry in the synthesis of tetrahydroquinolines, imidazoles, benzimidazole, and others.

https://orcid.org/0000-0003-1440-9221

César Augusto Sierra ^**e-mail: casierraa@unal.edu.co

Grupo de Investigación en Macromoléculas, Departamento de Química, Universidad Nacional de Colombia, Sede Bogotá, Carrera 45 26-85, A.A 5997, Bogotá, Colombia

Cesar A. Sierra studied chemistry at the Universidad Industrial de Santander-Colombia and obtained his PhD in 2005 with Prof Paul Lahti at the University of Massachusetts, Amherst. Upon returning to his native country, he established the research group on macromolecules, and currently acts as head of the Chemistry Department at the Universidad Nacional de Colombia. The implementation of nanotechnology in packaging, sensors, and textiles is within his current research interests, taking the results obtained until his incorporation in innovative Colombian companies, where he is exploring his facet as an entrepreneur.

*e-mail: casierraa@unal.edu.co

Mayra Beltrán-Pineda was born in Tunja-Boyacá, graduated as a Biologist from the Universidad Pedagógica y Tecnológica de Colombia in 2004. She was a young researcher at COLCIENCIAS in 2006. She obtained her Master’s degree in Sciences-Microbiology in 2009 from the Universidad Nacional de Colombia. Her research area is environmental microbiology, biotechnology, and nanotechnology. She is currently a PhD student in Biotechnology from the Universidad Nacional de Colombia under the supervision of Professor Cesar Sierra in the area of nanobiotechnology. Her doctoral research is focused on the biosynthesis of silver nanoparticles for the control of a potato plant pathogen.

Diana C. Peña-Solórzano was born in Calarcá, Quindío, and graduated in Chemistry from the Universidad Nacional of Colombia (2011). She obtained a double doctoral degree from the Universidad Nacional of Colombia and Regensburg University in Germany (2017). She held a postdoctoral degree at Universidad Nacional of Colombia (2019). Her research interests are in organic chemistry with applications in medicine, including the synthesis, characterization, and inhibitory activity evaluation of new analogs of tariquidar on breast cancer resistance protein (ABCG2). She is also interested in the development of eutectic solvents based on zinc chloride for green chemistry in the synthesis of tetrahydroquinolines, imidazoles, benzimidazole, and others.

Cesar A. Sierra studied chemistry at the Universidad Industrial de Santander-Colombia and obtained his PhD in 2005 with Prof Paul Lahti at the University of Massachusetts, Amherst. Upon returning to his native country, he established the research group on macromolecules, and currently acts as head of the Chemistry Department at the Universidad Nacional de Colombia. The implementation of nanotechnology in packaging, sensors, and textiles is within his current research interests, taking the results obtained until his incorporation in innovative Colombian companies, where he is exploring his facet as an entrepreneur.

Technique	Basis	Limit of detection/ (CFU mL⁻¹)	Advantages	Disadvantages	Reference
Polymerase chain reaction (PCR)	enzymatic amplification in vitro of a DNA fragment, carried out using two flanking oligonucleotides at the two ends of the target called primers DNA	10³-10⁴	standardized technology, easy operation	the effectiveness depends on the DNA extraction process, presence of inhibitors, polymerase activity, the concentration of the buffer and of DNTP	²2 Bej, A. K.; Mahbubani, M. H.; PCR Methods Appl. 1992, 1, 151.,³3 Fang, Y.; Ramasamy, R. P.; Biosensors 2015, 5, 537.
Fluorescent in situ hybridization (FISH)	hybridization of a DNA probe to its complementary sequence in previously fixed preparations; the probes are directly labeled with fluorescent nucleotides or indirectly by molecules that are subsequently detected by fluorescent antibodies; finally, the probes and target sequences are detected and visualized in situ through microscopic analysis	10³	high sensitivity	autofluorescence, photochemical destruction of the fluorophore	³3 Fang, Y.; Ramasamy, R. P.; Biosensors 2015, 5, 537.,⁴4 Volpi, E. V.; Bridger, J. M.; Biotechniques 2008, 45, 385.
Enzyme-linked immunoassay (ELISA)	detection with enzymes of antigenantibody binding; the enzyme converts a non-colored substrate into a colored product as an indicator of the binding event	10⁵-10⁶	low cost, the color change is used for detection	low sensitivity to detect bacteria	³3 Fang, Y.; Ramasamy, R. P.; Biosensors 2015, 5, 537.,⁵5 Ma, H.; Shieh, K.-J.; Lee, S.-L.; Nature 2006, 4, 36.
Immunofluorescence (IF)	detection of the presence of antibodies bound to antigens in tissues or body fluids, involving the emission of light from a substance that has been irradiated with a specific wavelength	10³	high sensitivity	photochemical destruction of the fluorophore	³3 Fang, Y.; Ramasamy, R. P.; Biosensors 2015, 5, 537.,⁶6 Mohan, K.; Pai, S.; Rao, R.; Sripathi, H.; Prabhu, S.; Indian J. Dermatol. Venereol. Leprol. 2008, 74, 415.
Flow cytometry (CMF)	a technique that allows the analysis of individual cells marked with Flow cytometry (CMF) fluorochromes, in which the detection mechanism involves light refraction	10⁴	simultaneous measurements of several parameters, fast detection	high price	³3 Fang, Y.; Ramasamy, R. P.; Biosensors 2015, 5, 537.,⁷7 Brown, M.; Wittwer, C.; Clin. Chem. 2000, 46, 1221.
Loop-mediated isothermal amplification (LAMP)	the reaction mixture is incubated at a specific temperature using a single tube Loop-mediated containing a buffer, target DNA, DNA isothermal amplification polymerase, and primers; (LAMP)the detection of the amplified product occurs by the presence of a precipitate	10⁷-10⁸	simple, easy to use, and rapid, performed at a constant temperature	complicated primer design; restricted availability of reagents and equipment in some countries; limitations for multiplexing	⁸8 Craw, P.; Balachandran, W.; Lab Chip 2012, 12, 2469; Kokkinos, P.; Ziros, P.; Bellou, M.; Vantarakis, A.; Food Anal. Methods 2014, 7, 512.

Name	Definition	Characteristics	Chemical precursors	Reference
Gold nanoparticles (AuNPs)	metallic nanoparticles with an affinity for sulfhydryl groups with which covalent bonds may be established	various shapes: spheres, cubes, hexagons, or bars; ability to scatter light; fluorescents: color change from blue to red; chemically stable; good aggregation; excellent conductivity; zero intrinsic toxicity; easily functionalized	tetrachloroaurate solution (0.1 mg mL⁻¹) and trisodium citrate (1%); hydrogen tetrachloroaurate(III) trihydrate aqueous solution (1 mM), sodium citrate (38.8 mmol L⁻¹); chloroauric acid (0.10 mol L⁻¹) aqueous solution; chloroauric acid (250 µmol L⁻¹), CTAB (100 mmol L⁻¹) and ascorbic acid (100 mol L⁻¹); dextran solution (1%) and chloroauric acid (0.01 mol L⁻¹); polydimethylsiloxane (PDMS), and hydrogen tetrachloroaurate(III) 1%; gold(III) chloride trihydrate solution (1%), and trisodium citrate solution (1%)	¹⁸18 Ho, K.-C.; Tsai, P.-J.; Lin, Y.-S.; Chen, Y.-C.; Anal. Chem. 2004, 76, 7162. 19 Nath, S.; Kaittanis, C.; Tinkham, A.; Perez, J. M.; Anal. Chem. 2008, 80, 1033. 20 Zhang, D.; Carr, D. J.; Alocilja, E. C.; Biosens. Bioelectron. 2009, 24, 1377. 21 Sahoo, A. K.; Sharma, S.; Chattopadhyay, A.; Ghosh, S. S.; Nanoscale 2012, 4, 1688. 22 Zhou, H.; Yang, D.; Ivleva, N. P.; Mircescu, N. E.; Niessner, R.; Haisch, C.; Anal. Chem. 2014, 86, 1525. 23 Tanvir, F.; Yaqub, A.; Tanvir, S.; Anderson, W. A.; Enzyme Microb. Technol. 2017, 99, 49.-²⁴24 Verma, P.; Maheshwari, S. K.; Int. J. Nano Dimens. 2019, 10, 18.
Silver nanoparticles (AgNPs)	metallic nanoparticles with sizes between 5 and 10 nm	different forms: spheres, bars or triangles; compared with the great majority of organic and inorganic chromophores, AgNPs have more efficient interaction with visible light	silver nitrate (0.12 mmol L⁻¹) and hydroxylamine hydrochloride (0.17 mmol L⁻¹); silver nitrate (0.018 mol L⁻¹), trisodium citrate (0.017 mol L⁻¹) and sodium borohydride (0.01 M); dry leaf extract, and silver nitrate (100 mmol L⁻¹); silver nitrate (5 × 10⁻³mol L⁻¹), sodium borohydride (2 × 10⁻³mol L⁻¹), and poly (vinyl alcohol) 1%; aqueous extract of Padina sp., and aqueous silver nitrate (0.01 mol L⁻¹)	²⁰20 Zhang, D.; Carr, D. J.; Alocilja, E. C.; Biosens. Bioelectron. 2009, 24, 1377.
Silica nanoparticles (SiNPs)	NPs constituted by a silica matrix and characterized by pores with a diameter between 2 and 50 nm	elevated surface area; modifiable pore volume; mechanical strength; chemical and thermal stability; biocompatibility; non-toxic; biodegradable; inert	TEOS and ammonium hydroxide; TEOS and ammonia solution (25 wt.%); 3-amino propyltrimethoxysilane, tetraethyl orthosilicate (1 mol L⁻¹), and ammonium hydroxide; cyclohexane, hexyl alcohol, Triton X-100, water, and TEOS; L-arginine water solution (6 mmol L⁻¹), TEOS, SiO₂, ethanol, and ammonium hydroxide	²⁵25 Thepwiwatjit, N.; Thattiyaphong, A.; Limsuwan, P.; Tuitemwong, K.; Tuitemwong, P.; J. Nanomater. 2013, 2013, 92.,²⁶26 Chitra, K.; Annadurai, G.; J. Nanotechnol. 2013, ID 509628.
Magnetic nanoparticles (MNPs)	iron oxide particles composed of a magnetite core	biocompatibility. easily biodegradable; functionalizable with amino and carboxyl groups; used to separate or enrich a target analyte	ferric chloride (FeCl₃, 4 mmol), urea (12 mmol), sodium citrate (8 mmol) and polyacrylamide (0.6 g); ferric chloride (FeCl₃), iron(II) chloride (FeCl₂) and aqueous hydrochloric acid (HCl, 2 mol L⁻¹); ferric chloride (FeCl₃, 1 mol L⁻¹), iron sulfate (II) (FeSO₄, 1 mol L⁻¹) and aqueous hydrochloric acid (HCl, 2 mol L⁻¹); iron(III) acetylacetonate and benzyl alcohol	¹⁰10 Gilmartin, N.; O’Kennedy, R.; Enzyme Microb. Technol. 2012, 50, 87.,¹²12 Kaittanis, C.; Santra, S.; Perez, J. M.; Adv. Drug Delivery Rev. 2010, 62, 408.,²⁷27 Lin, Y.-S.; Tsai, P.-J.; Weng, M.-F.; Chen, Y.-C.; Anal. Chem. 2005, 77, 1753.,²⁸28 Ileš, D.; Martinović, G.; Kozak, D.; Strojarstvo 2011, 53, 127.
Quantum dots (QDs)	particles formed by a semiconductor core material (mixtures of cadmium-selenium or cadmium-tellurium) covered by an additional semiconductor layer, usually zinc sulfide	colloidal and fluorescent semiconductors with unique optical properties; diameters of 2-8 nm; by coating with an organic material layer, surface functionalization for the binding of biomolecules is achieved; bright with high photostability	cadmium oxide (0.2 mol L⁻¹), selenium (2 mmol L⁻¹), sulfur solution (0.04 mol L⁻¹), trioctylphosphine oxide (0.5 g), tributylphosphine (0.472 g), 1-octadecene (2 g), oleic acid (1.5 g), octadecylamine (1.5 g) and stearic acid (0.8 mmol L⁻¹); cadmium selenide (CdSe) nanocrystals, and zinc sulfide (ZnS); mercaptoacetic acid liquid, aqueous CdCl₂ (0.01 mol L⁻¹), aqueous Na₂S (5 mmol L⁻¹), and chlorophyll	¹⁰10 Gilmartin, N.; O’Kennedy, R.; Enzyme Microb. Technol. 2012, 50, 87.,¹²12 Kaittanis, C.; Santra, S.; Perez, J. M.; Adv. Drug Delivery Rev. 2010, 62, 408.,²⁸28 Ileš, D.; Martinović, G.; Kozak, D.; Strojarstvo 2011, 53, 127.,²⁹29 Tully, E.; Hearty, S.; Leonard, P.; O’Kennedy, R.; Int. J. Biol. Macromol. 2006, 39, 127.
Carbon nanotubes	monoatomic layers of carbon atoms perfectly aligned and arranged in beehive structures wound in a cylindrical tube or several coupled tubes	sizes between 1 nm to a few microns or more; photoluminescent; semiconductors: excellent electrical properties; work as signal amplifiers and transducers	iron nitrate in isopropanol, 500 nm layer of silicon dioxide; graphite flakes, sulfuric acid, sodium nitrite, sodium permanganate, and deionized water; SWCNTs in DMF	¹⁰10 Gilmartin, N.; O’Kennedy, R.; Enzyme Microb. Technol. 2012, 50, 87.,¹²12 Kaittanis, C.; Santra, S.; Perez, J. M.; Adv. Drug Delivery Rev. 2010, 62, 408.,³⁰30 Dai, H.; Acc. Chem. Res. 2002, 35, 1035.
Metal-organic frameworks (MOFs)	two- or three-dimensional coordination networks formed by metal complexes linked to multifunctional organic ligands	high surface area; thermal/mechanical stability; good optical properties; resistance to photobleaching	Zn(NO₃)₂.6H₂O (16 mmol), 2-amino terephthalic acid (8 mmol), DMF (160 mL) and trimethylamine (64 mmol); Cu(NO₃)₂·3H₂O (0.015 mmol), trifluoroacetic acid (1.0 mol L⁻¹), and polyvinylpyrrolidone; ZrCl₄, DMF, and CuCl₂.2H₂O	¹³13 Bhardwaj, N.; Bhardwaj, S. K.; Nayak, M. K.; Mehta, J.; Kim, K.-H.; Deep, A.; TrAC, Trends Anal. Chem. 2017, 97, 120.,³¹31 Bon, V.; Senkovska, I.; Kaskel, S. In Nanoporous Materials for Gas Storage; Kaneko, K.; Rodríguez-Reinoso, F., eds.; Springer: Singapore, 2019, p. 137; Robin, A. Y.; Fromm, K. M.; Coord. Chem. Rev. 2006, 250, 2127; Stock, N.; Biswas, S.; Chem. Rev. 2011, 112, 933.

Type	Operating principle	Reference
Based on spectroscopy	using SRP (surface plasmon resonance) as a direct means to measure the binding of a target molecule to a surface ligand, light is reflected from the detection surface when binding events occur;this method measures reflected light changes as an interaction between the target molecule and receptors occurs on the detection surface; the SRP-based sensors detect the binding of an analyte to the bioreceptor by monitoring the change in the wavelength necessary to excite the SRP	²²22 Zhou, H.; Yang, D.; Ivleva, N. P.; Mircescu, N. E.; Niessner, R.; Haisch, C.; Anal. Chem. 2014, 86, 1525.,³⁷37 Mathelié-Guinlet, M.; Gammoudi, I.; Beven, L.; Moroté, F.; Delville, M.-H.; Grauby-Heywang, C.; Cohen-Bouhacina, T.; Procedia Eng. 2016, 168, 1048. 38 Tang, X.; Bansaruntip, S.; Nakayama, N.; Yenilmez, E.; Chang, Y.-l.; Wang, Q.; Nano Lett. 2006, 6, 1632. 39 Wen, C.-Y.; Hu, J.; Zhang, Z.-L.; Tian, Z.-Q.; Ou, G.-P.; Liao, Y.-L.; Li, Y.; Xie, M.; Sun, Z.-Y.; Pang, D.-W.; Anal. Chem. 2013, 85, 1223. 40 Chen, C.; Wang, Q.; Am. J. Nano Res. Appl. 2015, 3, 13. 41 Chattopadhyay, S.; Sabharwal, P. K.; Jain, S.; Kaur, A.; Singh, H.; Anal. Chim. Acta 2019, 1067, 98.-⁴²42 Dong, X.; Shi, Z.; Xu, C.; Yang, C.; Chen, F.; Lei, M.; Wang, J.; Cui, Q.; Biosens. Bioelectron. 2020, 149, 111843.
Optical	usually based on semiconductor particles that fluoresce when excited at different wavelengths by a light source;when the analyte of interest (yellow sphere) interacts with the sensor, fluorescence is emitted due to the semiconductor particles’ excitation; an optical detection unit captures the fluorescence that evidences the union	¹⁴14 Zhao, X.; Hilliard, L. R.; Mechery, S. J.; Wang, Y.; Bagwe, R. P.; Jin, S.; Tan, W.; Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 15027.,¹⁸18 Ho, K.-C.; Tsai, P.-J.; Lin, Y.-S.; Chen, Y.-C.; Anal. Chem. 2004, 76, 7162.,¹⁹19 Nath, S.; Kaittanis, C.; Tinkham, A.; Perez, J. M.; Anal. Chem. 2008, 80, 1033., ²⁵25 Thepwiwatjit, N.; Thattiyaphong, A.; Limsuwan, P.; Tuitemwong, K.; Tuitemwong, P.; J. Nanomater. 2013, 2013, 92. 26 Chitra, K.; Annadurai, G.; J. Nanotechnol. 2013, ID 509628.-²⁷27 Lin, Y.-S.; Tsai, P.-J.; Weng, M.-F.; Chen, Y.-C.; Anal. Chem. 2005, 77, 1753.,⁴¹41 Chattopadhyay, S.; Sabharwal, P. K.; Jain, S.; Kaur, A.; Singh, H.; Anal. Chim. Acta 2019, 1067, 98.,⁴³43 El-Boubbou, K.; Gruden, C.; Huang, X.; J. Am. Chem. Soc. 2007, 129, 13392. 44 Dogra, N.; Li, X.; Kohli, P.; Langmuir 2012, 28, 12989. 45 Phillips, R. L.; Miranda, O. R.; You, C. C.; Rotello, V. M.; Bunz, U. H.; Angew. Chem. 2008, 47, 2590. 46 Villamizar, R. A.; Maroto, A.; Rius, F. X.; Inza, I.; Figueras, M. J.; Biosens. Bioelectron. 2008, 24, 279. 47 Abdeen, S.; Isaac, R. R.; Geo, S.; Sornalekshmi, S.; Rose, A.; Praseetha, P.; Nano Biomed. Eng. 2013, 5, 39; Alghuthaymi, M. A.; Almoammar, H.; Rai, M.; Said-Galiev, E.; Abd-Elsalam, K. A.; Biotechnol. Biotechnol. Equip. 2015, 29, 221. 48 Kumar, P.; Deep, A.; Kim, K.-H.; TrAC, Trends Anal. Chem. 2015, 73, 39. 49 Zhang, D.; Zhou, Y.; Cuan, J.; Gan, N.; CrystEngComm 2018, 20, 1264. 50 Ardekani, S. M.; Dehghani, A.; Ye, P.; Nguyen, K.-A.; Gomes, V. G.; J. Colloid Interface Sci. 2019, 552, 378. 51 Mathelié-Guinlet, M.; Cohen-Bouhacina, T.; Gammoudi, I.; Martin, A.; Béven, L.; Delville, M.-H.; Grauby-Heywang, C.; Sens. Actuators, B 2019, 292, 314. 52 Mou, X.-Z.; Chen, X.-Y.; Wang, J.; Zhang, Z.; Yang, Y.; Shou, Z.-X.; Tu, Y.-X.; Du, X.; Wu, C.; Zhao, Y.; ACS Appl. Mater. Interfaces 2019, 11, 23093. 53 Suaifan, G. A.; Zourob, M.; Microchim. Acta 2019, 186, 230. 54 Yu, S.; Tang, Y.; Yan, M.; Aguilar, Z. P.; Lai, W.; Xu, H.; Sens. Actuators, B 2019, 279, 31.-⁵⁵55 Murasova, P.; Kovarova, A.; Kasparova, J.; Brozkova, I.; Hamiot, A.; Pekarkova, J.; Dupuy, B.; Drbohlavova, J.; Bilkova, Z.; Korecka, L.; J. Electroanal. Chem. 2020, 114051.
Mechanics	based on atomic force microscopy: high sensitivity requires small sample volumes for which no marking is necessary; this technique uses a nanometer-sized tip-shaped structure to scan the surface to which the pathogen binds; then, a detailed map of the surface is generated, revealing the presence of the agent; this technique has the advantage of allowing the simultaneous scanning of several pathogens microcantilevers: biosensors in the form of scaffolds with a section for selective recognition of either antibody-antigens or DNA fragments with their complementary probes; the analytes bind to the device in a dynamic manner in which the binding of the target molecule generates a change in the resonance frequency or in a static way where the deflection of the scaffold occurs by the binding of the target molecule	⁴⁴44 Dogra, N.; Li, X.; Kohli, P.; Langmuir 2012, 28, 12989.,⁵²52 Mou, X.-Z.; Chen, X.-Y.; Wang, J.; Zhang, Z.; Yang, Y.; Shou, Z.-X.; Tu, Y.-X.; Du, X.; Wu, C.; Zhao, Y.; ACS Appl. Mater. Interfaces 2019, 11, 23093.,⁵³53 Suaifan, G. A.; Zourob, M.; Microchim. Acta 2019, 186, 230.,⁵⁶56 Huang, Y.-F.; Wang, Y.-F.; Yan, X.-P.; Environ. Sci. Technol. 2010, 44, 7908.

Bacterial species	Reference
Escherichia coli	¹⁴14 Zhao, X.; Hilliard, L. R.; Mechery, S. J.; Wang, Y.; Bagwe, R. P.; Jin, S.; Tan, W.; Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 15027.,¹⁹19 Nath, S.; Kaittanis, C.; Tinkham, A.; Perez, J. M.; Anal. Chem. 2008, 80, 1033.,²⁶26 Chitra, K.; Annadurai, G.; J. Nanotechnol. 2013, ID 509628.,⁴³43 El-Boubbou, K.; Gruden, C.; Huang, X.; J. Am. Chem. Soc. 2007, 129, 13392.,⁴⁴44 Dogra, N.; Li, X.; Kohli, P.; Langmuir 2012, 28, 12989.,⁵⁷57 Edgar, R.; McKinstry, M.; Hwang, J.; Oppenheim, A. B.; Fekete, R. A.; Giulian, G.; Merril, C.; Nagashima, K.; Adhya, S.; Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 4841. 58 Mukhopadhyay, B.; Martins, M. B.; Karamanska, R.; Russell, D. A.; Field, R. A.; Tetrahedron Lett. 2009, 50, 886. 59 Guven, B.; Basaran-Akgul, N.; Temur, E.; Tamer, U.; Boyacı, İ. H.; Analyst 2011, 136, 740. 60 Maurer, E. I.; Comfort, K. K.; Hussain, S. M.; Schlager, J. J.; Mukhopadhyay, S. M.; Sensors 2012, 12, 8135. 61 Gasparyan, V. K.; Bazukyan, I. L.; Anal. Chim. Acta 2013, 766, 83. 62 Petaccia, M.; Bombelli, C.; Paroni Sterbini, F.; Papi, M.; Giansanti, L.; Bugli, F.; Sanguinetti, M.; Mancini, G.; Sens. Actuators, B 2017, 248, 247. 63 Villamizar, R. A.; Maroto, A.; Rius, F. X.; Sens. Actuators, B 2009, 136, 451. 64 García-Aljaro, C.; Cella, L. N.; Shirale, D. J.; Park, M.; Muñoz, F. J.; Yates, M. V.; Mulchandani, A.; Biosens. Bioelectron. 2010, 26, 1437. 65 Chen, L.; Zhang, J.; J. Biosens. Bioelectron. 2012, S11, 005. 66 Thuy, N. T.; Tam, P. D.; Tuan, M. A.; Le, A.-T.; Van Thu, V.; Van Hieu, N.; Chien, N. D.; Curr. Appl. Phys. 2012, 12, 1553. 67 Bhardwaj, N.; Bhardwaj, S. K.; Mehta, J.; Nayak, M. K.; Deep, A.; New J. Chem. 2016, 40, 8068. 68 Muniandy, S.; Teh, S. J.; Thong, K. L.; Thiha, A.; Dinshaw, I. J.; Lai, C. W.; Ibrahim, F.; Leo, B. F.; Crit. Rev. Anal. Chem. 2019, 49, 510. 69 Qiu, Q.; Chen, H.; Ying, S.; Sharif, S.; You, Z.; Wang, Y.; Ying, Y.; Microchim. Acta 2019, 186, 93.-⁷⁰70 You, Z.; Qiu, Q.; Chen, H.; Feng, Y.; Wang, X.; Wang, Y.; Ying, Y.; Biosens. Bioelectron. 2020, 150, 111896.
Staphylococcus aureus	⁷¹71 Wang, B.; Huang, X.; Ma, M.; Shi, Q.; Cai, Z.; Food Control. 2014, 35, 26. 72 Shahrokhian, S.; Ranjbar, S.; Analyst 2018, 143, 3191.-⁷³73 Sobhan, A.; Lee, J.; Park, M.-K.; Oh, J.-H.; LWT 2019, 108, 48.
Staphylococcus saprophyticus	¹⁸18 Ho, K.-C.; Tsai, P.-J.; Lin, Y.-S.; Chen, Y.-C.; Anal. Chem. 2004, 76, 7162.
Mycobacterium tuberculosis	⁷⁴74 Chen, X.; Tang, M.; Liu, Y.; Huang, J.; Liu, Z.; Tian, H.; Zheng, Y.; de la Chapelle, M. L.; Zhang, Y.; Fu, W.; Microchim. Acta 2019, 186, 102.
Salmonella typhimurium	⁷⁵75 Carrillo-Carrión, C.; Simonet, B. M.; Valcárcel, M.; Biosens. Bioelectron. 2011, 26, 4368. 76 Chunglok, W.; Wuragil, D. K.; Oaew, S.; Somasundrum, M.; Surareungchai, W.; Biosens. Bioelectron. 2011, 26, 3584. 77 Joo, J.; Yim, C.; Kwon, D.; Lee, J.; Shin, H. H.; Cha, H. J.; Jeon, S.; Analyst 2012, 137, 3609. 78 Zelada-Guillén, G. A.; Sebastián-Avila, J. L.; Blondeau, P.; Riu, J.; Rius, F. X.; Biosens. Bioelectron. 2012, 31, 226. 79 Yaohua, H.; Chengcheng, W.; Bing, B.; Mintong, L.; Wang, R.; Li, Y.; Int. J. Agric. Biol. Eng. 2014, 7, 77.-⁸⁰80 Xue, L.; Huang, F.; Hao, L.; Cai, G.; Zheng, L.; Li, Y.; Lin, J.; Food Chem. 2020, 126719.
Brucella abortus	⁵⁶56 Huang, Y.-F.; Wang, Y.-F.; Yan, X.-P.; Environ. Sci. Technol. 2010, 44, 7908.
Salmonella sp.	⁴¹41 Chattopadhyay, S.; Sabharwal, P. K.; Jain, S.; Kaur, A.; Singh, H.; Anal. Chim. Acta 2019, 1067, 98.,⁵⁹59 Guven, B.; Basaran-Akgul, N.; Temur, E.; Tamer, U.; Boyacı, İ. H.; Analyst 2011, 136, 740.,⁷⁷77 Joo, J.; Yim, C.; Kwon, D.; Lee, J.; Shin, H. H.; Cha, H. J.; Jeon, S.; Analyst 2012, 137, 3609.,⁸⁰80 Xue, L.; Huang, F.; Hao, L.; Cai, G.; Zheng, L.; Li, Y.; Lin, J.; Food Chem. 2020, 126719.
Salmonella enterica	²³23 Tanvir, F.; Yaqub, A.; Tanvir, S.; Anderson, W. A.; Enzyme Microb. Technol. 2017, 99, 49.,³⁸38 Tang, X.; Bansaruntip, S.; Nakayama, N.; Yenilmez, E.; Chang, Y.-l.; Wang, Q.; Nano Lett. 2006, 6, 1632.,⁴⁵45 Phillips, R. L.; Miranda, O. R.; You, C. C.; Rotello, V. M.; Bunz, U. H.; Angew. Chem. 2008, 47, 2590.,⁸¹81 Zhong, M.; Yang, L.; Yang, H.; Cheng, C.; Deng, W.; Tan, Y.; Xie, Q.; Yao, S.; Biosens. Bioelectron. 2019, 126, 493.
Pseudomonas aeruginosa	³⁸38 Tang, X.; Bansaruntip, S.; Nakayama, N.; Yenilmez, E.; Chang, Y.-l.; Wang, Q.; Nano Lett. 2006, 6, 1632.,³⁹39 Wen, C.-Y.; Hu, J.; Zhang, Z.-L.; Tian, Z.-Q.; Ou, G.-P.; Liao, Y.-L.; Li, Y.; Xie, M.; Sun, Z.-Y.; Pang, D.-W.; Anal. Chem. 2013, 85, 1223.,⁸²82 Wu, S.; Duan, N.; Shen, M.; Wang, J.; Wang, Z.; Microchim. Acta 2019, 186, 401. 83 Banasiuk, R.; Krychowiak, M.; Swigon, D.; Tomaszewicz, W.; Michalak, A.; Chylewska, A.; Ziabka, M.; Lapinski, M.; Koscielska, B.; Narajczyk, M.; Arabian J. Chem. 2020, 13, 1415.-⁸⁴84 Wang, Q.-Y.; Kang, Y.-J.; Nanoscale Res. Lett. 2016, 11, 150.
Bacillus subtilis	⁴⁹49 Zhang, D.; Zhou, Y.; Cuan, J.; Gan, N.; CrystEngComm 2018, 20, 1264.,⁶¹61 Gasparyan, V. K.; Bazukyan, I. L.; Anal. Chim. Acta 2013, 766, 83.,⁸⁵85 Zhang, X.; Xie, G.; Gou, D.; Luo, P.; Yao, Y.; Chen, H.; Biosens. Bioelectron. 2019, 142, 111486.
Bacillus cereus	⁵⁹59 Guven, B.; Basaran-Akgul, N.; Temur, E.; Tamer, U.; Boyacı, İ. H.; Analyst 2011, 136, 740.,⁸⁶86 Sun, J.; Ji, J.; Sun, Y.; Abdalhai, M. H.; Zhang, Y.; Sun, X.; Biosens. Bioelectron. 2015, 70, 239.
Candida albicans	⁸⁷87 Liu, F.; Choi, K. S.; Park, T. J.; Lee, S. Y.; Seo, T. S.; BioChip J. 2011, 5, 123.
Enterococcus faecalis	⁶²62 Petaccia, M.; Bombelli, C.; Paroni Sterbini, F.; Papi, M.; Giansanti, L.; Bugli, F.; Sanguinetti, M.; Mancini, G.; Sens. Actuators, B 2017, 248, 247.
Vibrio cholerae	²⁷27 Lin, Y.-S.; Tsai, P.-J.; Weng, M.-F.; Chen, Y.-C.; Anal. Chem. 2005, 77, 1753.,⁸⁸88 Gupta, A.; Bhardwaj, S. K.; Sharma, A. L.; Kim, K.-H.; Deep, A.; Environ. Res. 2019, 171, 395.
Cryptosporidium parvum	⁸⁹89 Zhu, L.; Ang, S.; Liu, W.-T.; Appl. Environ. Microbiol. 2004, 70, 597.
Giardia lamblia	⁸⁹89 Zhu, L.; Ang, S.; Liu, W.-T.; Appl. Environ. Microbiol. 2004, 70, 597.

Functionalized substances	Pathogens or analyte to detect	Operating principle	Limit of detection	Detection time / h	Reference
Antibody immunoglobuline G (IgG) anti-SEB	enterotoxin Bfrom Staphylococcus aureus	on this lamella specific antibodies (anti-SEB IgG) are functionalized, binding to the protein’s carboxyl group; after contact with the analyte, the affinity interaction takes place, but a process of amplification of the signal is required; for that, two additional antibodies are used to detect very low toxin concentrations	10⁻⁵mol	0.2	⁹¹91 Naimushin, A. N.; Soelberg, S. D.; Nguyen, D. K.; Dunlap, L.; Bartholomew, D.; Elkind, J.; Melendez, J.; Furlong, C. E.; Biosens. Bioelectron. 2002, 17, 573.
Immunoglobuline G (IgG)	Staphylococcus aureus, S. saprophyticus	when magnetic nanoparticles functionalized with IgG are used, dispersed bacterial cells are selectively concentrated in a solution facilitating their detection	ND	1	¹⁸18 Ho, K.-C.; Tsai, P.-J.; Lin, Y.-S.; Chen, Y.-C.; Anal. Chem. 2004, 76, 7162.
Poly(p-phenylene ethynylene) (PPE)	Amycolatopsis azurea, Amycolatopsis orientalis subsp.	the interaction of bacteria with the nanoparticle-PPE conjugate produces the release of anionic PPE, which restores fluorescence	ND	0.5	⁴⁵45 Phillips, R. L.; Miranda, O. R.; You, C. C.; Rotello, V. M.; Bunz, U. H.; Angew. Chem. 2008, 47, 2590.
DNA	Salmonella enterica	there is an exponential relationship between the target DNA concentration and the released barcode DNA’s fluorescence signal	1 ng mL⁻¹	ND	²⁰20 Zhang, D.; Carr, D. J.; Alocilja, E. C.; Biosens. Bioelectron. 2009, 24, 1377.
Thiolated RNA probe	Escherichia coli	carbon nanotubes coated with AuNPs functionalized with a thiolated RNA probe-specific (aptamers) can bind efficiently to Escherichia coli, which is evidenced by a change in absorbance	ND	0.3	⁶⁰60 Maurer, E. I.; Comfort, K. K.; Hussain, S. M.; Schlager, J. J.; Mukhopadhyay, S. M.; Sensors 2012, 12, 8135.
Paracetamol dimer (PD)	Pediococcus acidilactici Bacillus cereus	fluorescent nanocomposite, consisting of paracetamol dimer (PD) and AuNPs, successfully detects six bacterial strains; the Gram-positive and Gram-negative bacteria quench the fluorescence	100 CFU mL⁻¹	12	²¹21 Sahoo, A. K.; Sharma, S.; Chattopadhyay, A.; Ghosh, S. S.; Nanoscale 2012, 4, 1688.
DNA	Escherichia coli O157:H7	a fluorescence “on/off” switch to detect E. coli O157:H7 was created, the stem-loop DNA probe reached the maximum fluorescence intensity value when there was 10 nm between Cy3 and the surface of the Au@Ag nanorods	3.33 × 10⁻¹⁸mol L⁻¹	7	⁸⁶86 Sun, J.; Ji, J.; Sun, Y.; Abdalhai, M. H.; Zhang, Y.; Sun, X.; Biosens. Bioelectron. 2015, 70, 239.
CTAB	Staphylococcus aureus, Escherichia coli, Salmonella enterica	the positively charged AuNPs bind to the negatively charged enzyme β-galactosidase causing its deactivation; the color change of the solution evidences the binding due to the enzymatic decomposition of the fluorogenic chlorophenol red β-D-galactopyranoside	10 CFU mL⁻¹	1	²³23 Tanvir, F.; Yaqub, A.; Tanvir, S.; Anderson, W. A.; Enzyme Microb. Technol. 2017, 99, 49.
Thiolated protein G (PrG-thiol)	Escherichia coli O157:H7	PrG is commonly used to immobilize antibodies on solid supports; when binding to immunoglobulin G (IgG), PrG leaves fragments of the antigen (Fab) aligned and with little steric hindrance, facilitating detection	48 CFU mL⁻¹	8	⁹²92 Lin, D.; Pillai, R. G.; Lee, W. E.; Jemere, A. B.; Microchim. Acta 2019, 186, 169.
Solid-phase isothermal recombinase polymerase amplification (RPA)	Citrus tristeza virus(CTV)	the CTV target amplified in situ was investigated by EIS in a redox system using an optimized RPA that includes an AuNP-modified electrode with a thiolated primer	1000 fg mL⁻¹	2	⁹³93 Khater, M.; Escosura-Muñiz, A.; Altet, L.; Merkoçi, A.; Anal. Chem. 2019, 91, 4790.
4-Mercaptobenzoic acid	Vibrio parahaemolyticus	detection measured by the decrease in the Raman signal at 1592 cm⁻¹, due to the electrostatic interaction between a negatively charged signaling probe (4-mercaptobenzoic acid bound to AuNP modified with aptamers) and cysteamine	12 CFU mL⁻¹	0.8	⁸²82 Wu, S.; Duan, N.; Shen, M.; Wang, J.; Wang, Z.; Microchim. Acta 2019, 186, 401.
Azide and alkyne functional molecules	Escherichia coli	possible by using the click reaction between modified AuNPs and azide and alkyne functional molecules, catalyzed by Cu⁺, producing in the solution a change from red to blue due to the aggregation of nanoparticles	40 CFU mL⁻¹	1	⁵²52 Mou, X.-Z.; Chen, X.-Y.; Wang, J.; Zhang, Z.; Yang, Y.; Shou, Z.-X.; Tu, Y.-X.; Du, X.; Wu, C.; Zhao, Y.; ACS Appl. Mater. Interfaces 2019, 11, 23093.
Fullerene nanoparticles/ nitrogen-doped graphene (nano-C60/NGS)	Mycobacterium tuberculosis	Au-nano-C60/NGS acting as a new signal tag produce a response in the absence of redox molecules; subsequently, to achieve signal amplification, it is labeled with signal probes creating a tracer label	3 fmol L⁻¹	ND	⁹⁴94 Bai, L.; Chen, Y.; Liu, X.; Zhou, J.; Cao, J.; Hou, L.; Guo, S.; Anal. Chim. Acta 2019, 1080, 75.
Polyaniline (PANI)	Pseudomonas aeruginosa	the PANI/AuNP prepared show to be a highly sensitive pyocyanin sensor in a pathogen culture, which uses electron microscopy and cyclic voltammetry to study morphology and electrical conductivity of the constructed electrode	500 nmol L⁻¹	24	⁹⁵95 Elkhawaga, A. A.; Khalifa, M. M.; El-Badawy, O.; Hassan, M. A.; El-Said, W. A.; PloS One 2019, 14, e0216438.
Oligonucleotide primers	Listeria monocytogenes, S. typhimurium, Escherichia coli	flower-shaped AuNPs were used as a colorimetric sensor; the detection of PCR products was visible to the naked eye by directly mixing them with FAuNPs and NaCl	10, 10 and 50 pg mL⁻¹	0.2	⁹⁶96 Du, J.; Wu, S.; Niu, L.; Li, J.; Zhao, D.; Bai, Y.; Anal. Methods 2020, 12, 212.
AuNPs	Escherichia coli (MTCC723 and MTCC443)	the growth of bacteria inhibition by ZnO-NPs is possible using fluorescence microscopy; due to changes in cell membrane permeability and intracellular metabolic system	ND	ND	⁹⁷97 Satpathy, G.; Chandra, G. K.; Manikandan, E.; Mahapatra, D. R.; Umapathy, S.; Biotechnol. Lett. 2020, 42, 853.
Perfluorodecanethiol (PFDT)	A/H1N1	release of Cy3-tagged aptamer DNA from nanopopcorn substrate surfaces through interaction between aptamer DNA-A/H1N1 virus was measured using the decrease in intensity of the resulting Raman peak	97 PFU mL⁻¹	0.3	⁹⁸98 Chen, H.; Park, S.-G.; Choi, N.; Moon, J.-I.; Dang, H.; Das, A.; Lee, S.; Kim, D.-G.; Chen, L.; Choo, J.; Biosens. Bioelectron. 2020, 167, 112496.

Functionalized substance	Pathogens or analyte to detect	Operating principle	Limit of detection	Detection time / h	Reference
Polyvinylpyrrolidone	Staphylococcus aureus Pseudomonas aeruginosa	this biosensor showed that the synthesis method (microwave or thermal radiation) and the composition of the plant extract have a strong influence on the nanoparticles’ activity; the antioxidant potential of the plant extract is critical for the antimicrobial activity of NPs and the reduction method applied to the metal	10 µg mL⁻¹	24	⁸³83 Banasiuk, R.; Krychowiak, M.; Swigon, D.; Tomaszewicz, W.; Michalak, A.; Chylewska, A.; Ziabka, M.; Lapinski, M.; Koscielska, B.; Narajczyk, M.; Arabian J. Chem. 2020, 13, 1415.
Urease	Salmonella	in the presence of harmful pathogens, the interaction between the receptor-coated AgNP and the bacterial surface is favored, causing urease to catalytically raise the pH of the solution, which allows detection by means of a pH-sensitive chromogenic dye	10 cells mL⁻¹	ND	⁹⁹99 Singh, P.; Kakkar, S.; Kumar, R.; Bhalla, V.; Chem. Commun. 2019, 55, 4765.
Piper betle (Pb)	Pseudomonas aeruginosa Chromobacterium violaceum	molecular docking of eugenol-conjugated AgNPs on quorum sensing regulatory proteins is followed by FTIR analysis, demonstrating that the phytochemical components of Pb had blocked Pb-AgNPs; this indicates that AgNP-eugenol system exhibits considerable binding interactions with QS-associated proteins	12.5, 25 µg mL⁻¹	20	¹⁰⁰100 Shah, S.; Gaikwad, S.; Nagar, S.; Kulshrestha, S.; Vaidya, V.; Nawani, N.; Pawar, S.; Biofouling 2019, 35, 34.

Functionalized substances	Pathogens or analyte to detect	Operating principle	Limit of detection	Detection time / min	Reference
Antibody	Escherichia coli	the bioconjugated nanoparticle can be followed by an extremely high fluorescent signal, giving it molecular biorecognition properties; then, allowing fast and accurate identification of a variety of bacteria	1-400 cells	20	¹⁴14 Zhao, X.; Hilliard, L. R.; Mechery, S. J.; Wang, Y.; Bagwe, R. P.; Jin, S.; Tan, W.; Proc. Natl. Acad. Sci. U. S. A. 2004, 101, 15027.
Dioxetane-polymyxin B	Escherichia coli	the reaction of β-galactosidase enzyme from E. coli with the dioxetane substrate generated light at 530 nm; the light emission for the porous silicon biosensor chip with E. coli was significantly more intense than that obtained for the control system, pristine and flat silicon chip	10¹ CFU	30	¹⁰²102 Mathew, F. P.; Alocilja, E. C.; Biosens. Bioelectron. 2005, 20, 1656.
D-Mannose	Escherichia coli	an external magnetic field is applied to separate the nanoparticles bound to the lectin from the bacterial surface stained with a fluorescent dye (Picogreen), a process recorded by fluorescence microscopy	10⁴cells mL⁻¹	5	⁴³43 El-Boubbou, K.; Gruden, C.; Huang, X.; J. Am. Chem. Soc. 2007, 129, 13392.
Carboxyl group and monoclonal antibody	Vibrio cholerae	SiNPs functionalized with carboxyl groups and conjugated with monoclonal antibodies specific for Vibrio cholerae are used as the amplification signal to detect this bacteria using fluorescence microscopy	1 cell	5	²⁵25 Thepwiwatjit, N.; Thattiyaphong, A.; Limsuwan, P.; Tuitemwong, K.; Tuitemwong, P.; J. Nanomater. 2013, 2013, 92.
Antibody	Salmonella typhimurium	the fluorescence intensity of S. typhimurium incubated with the anti-S. typhimurium antibody modified Ru(bpy)-doped fluorescent NP was measured by fluorescence microscopy; the fluorescence response is proportional to the quantity of S. typhimurium	110 CFU mL⁻¹	60	¹⁰³103 He, X.; Hu, C.; Guo, Q.; Wang, K.; Li, Y.; Shangguan, J.; Biosens. Bioelectron. 2013, 42, 460.
Antibody	Salmonella and Escherichia coli O157	SiNPs doped with fluorescein isothiocyanate and Ru(bpy), conjugated to the respective antibodies and used in a conventional lateral flow immunoassay, were followed using a nitrocellulose strip analyzed by a smartphone-based fluorimeter	10⁵CFU mL⁻¹	10	¹⁰⁴104 Rajendran, V. K.; Bakthavathsalam, P.; Ali, B. M. J.; Microchim. Acta 2014, 181, 1815.
Acridine orange	Staphylococcus aureus	acridine Orange@SiO₂nanoparticles dispersed with glutaraldehyde and incubated with S. aureus were detected by fluorescence spectrophotometry	500 CFU mL⁻¹	20	¹⁰⁵105 Jiang, Y.; Xu, Y.; Wang, R.; Zhao, B.; Zhang, X.; Zhao, H.; RSC Adv. 2015, 5, 54338.
Positive (PAH) and negative (PSS) polyelectrolytes	Escherichia coli	by cyclic voltammetry and QCM-D measurements, the detection of Escherichia coli is possible by tracking oxidation-reduction reactions and frequency changes, respectively	ND	2-4	³⁷37 Mathelié-Guinlet, M.; Gammoudi, I.; Beven, L.; Moroté, F.; Delville, M.-H.; Grauby-Heywang, C.; Cohen-Bouhacina, T.; Procedia Eng. 2016, 168, 1048.
DNA	Salmonella typhimurium	streptavidin conjugated to the surface of COOH-FSiNPs (tris(2,2’-bipyridyl)dichlororuthenium(II) hexahydrate doped with carboxyl modified silica NPs) is incubated with an aptamer labeled with specific ssDNA biotin; this aptamer-bacteria system is treated with synthetic streptavidin-conjugated silica nanoprobes (SA-FSiNP) for the detection by fluorescence of S. typhimurium	ND	10	⁸⁴84 Wang, Q.-Y.; Kang, Y.-J.; Nanoscale Res. Lett. 2016, 11, 150.
Polyelectrolyte multilayer (PEM)	Escherichia coli	functionalization applied to an electrochemical transducer for the detection of Escherichia coli	10⁶-10³CFU mL⁻¹	5-30	⁵¹51 Mathelié-Guinlet, M.; Cohen-Bouhacina, T.; Gammoudi, I.; Martin, A.; Béven, L.; Delville, M.-H.; Grauby-Heywang, C.; Sens. Actuators, B 2019, 292, 314.
Polyclonal antibody	Brucella abortus	the addition of conjugates to the bacterial suspension forms PMNP sandwich structures of Brucella abortus-blue-SiNPs (possible to separate by a magnet), releasing a blue tint measurable by absorbance at 670 nm	450 CFU mL⁻¹	60	¹⁰⁶106 Shams, A.; Rahimian Zarif, B.; Salouti, M.; Shapouri, R.; Mirzaii, S.; Artif. Cells, Nanomed., Biotechnol. 2019, 47, 2562.
Hemin (chemiluminescent (CL) tag molecule)	Escherichia coli O157:H7 Staphylococcus aureus	CL was encapsulated in silica NPs with ordered mesopores; DNA can specifically change upon exposure to DNA nuclease or bacterial lysate through the use of a specific DNA gate, resulting in a more significant release of encapsulated hemin and resulting in an improved CL signal for the luminol-H₂O₂system	3.0, 2.5 CFU mL⁻¹	60	¹⁰⁷107 Gu, Z.; Fu, A.; Ye, L.; Kuerban, K.; Wang, Y.; Cao, Z.; ACS Sens. 2019, 4, 2922.
Organic dyes (dark blue, reaction green, reaction Pseudomonas aeruginosa blue, bright red, and reactive purple)	Pseudomonas aeruginosa	monodisperse-colored SiNPs as agglutination test vehicles were prepared using reverse microemulsion; the SiNPs were sensitized with eleven types of monospecific antibodies against Pseudomonas aeruginosa (IgG-colored SiNPs); the agglutination test was able to identify different serotypes of P. aeruginosa	10⁹CFU mL⁻¹	1-3	¹⁰⁸108 Dong, X.-m.; Jing, T.; Li, T.-t.; Zhang, P.; Yan, Z.; Yu, T.; Su, R.-h.; Shi, D.-f.; J. Integr. Agric. 2019, 18, 1936.

Functionalized substance	Pathogens or analyte to detect	Operating principle	Limit of detection	Detection time / min	Reference
Nanoparticle-based proteomicstrategies	Escherichia coli O157:H7	a method based on tryptic digestion of bacteria captured under magnetic NP microwave irradiation of Fe₃O₄, followed by matrix-assisted laser ionization/desorption mass spectrometry analysis	30 cells mL⁻¹	1.5	⁹⁰90 Chen, W.-J.; Tsai, P.-J.; Chen, Y.-C.; Anal. Chem. 2008, 80, 9612.
Peptide	Escherichia coli O157:H7	biosensor based on the ability of E. coli O157:H7 proteases to modify their optical response; the gradual increase in the sensor surface’s golden hue after proteolysis can be correlated with the concentration of E. coli O157:H7 in the sample	12 CFU mL⁻¹	ND	¹¹⁰110 Suaifan, G. A.; Alhogail, S.; Zourob, M.; Biosens. Bioelectron. 2017, 92, 702.
Amine-functionalized magnetic nanoparticles (AFMNPs)	Escherichia coli	the positive charges on the surface of unmodified AFMNP promote strong electrostatic interactions with negatively charged sites on the surface of bacterial pathogens	ND	10	⁵⁶56 Huang, Y.-F.; Wang, Y.-F.; Yan, X.-P.; Environ. Sci. Technol. 2010, 44, 7908.
Antibody	Escherichia coli	the immobilization of biotin-labeled anti-Escherichia coli antibodies on avidin-coated magnetic nanoparticles allowed the synthesis of gold-coated magnetic NPs, a biosensor capable of separating and quantifying Escherichia coli	8 CFU mL⁻¹	70	⁵⁹59 Guven, B.; Basaran-Akgul, N.; Temur, E.; Tamer, U.; Boyacı, İ. H.; Analyst 2011, 136, 740.
Gentamicin (Gm)	Staphyloccocus aureus	the combination of a magnetic nucleus (Fe₃O₄), a fluorescent layer (SiO₂), and the conjugation with Gm, known as Gm-MNP, allows the capture of S. aureus cells through magnetic interaction with the Gm-MNP system	500 CFU mL⁻¹	1	⁶⁵65 Chen, L.; Zhang, J.; J. Biosens. Bioelectron. 2012, S11, 005.
Immunofluorescent nanospheres (IFNS)	Salmonella typhimurium	the combination of immunomagnetic nanospheres and IFNS allows the capture and specific recognition of S. typhimurium; the sandwich-type immune complex (IMNS-bacteria-IFNS) formed is easily analyzed by a fluorescence microscope	10 CFU mL⁻¹	ND	³⁹39 Wen, C.-Y.; Hu, J.; Zhang, Z.-L.; Tian, Z.-Q.; Ou, G.-P.; Liao, Y.-L.; Li, Y.; Xie, M.; Sun, Z.-Y.; Pang, D.-W.; Anal. Chem. 2013, 85, 1223.
Biotin-probe labeled magnetic beads (MBs)	Salmonella sp.	the primers were designed to produce MBs-ssDNA; using Fe₃O₄ NPs, the target ssDNA is isolated, which hybridized with the reporter probes can decrease signal	74 CFU mL⁻¹	80	⁵⁴54 Yu, S.; Tang, Y.; Yan, M.; Aguilar, Z. P.; Lai, W.; Xu, H.; Sens. Actuators, B 2019, 279, 31.
Streptavidin	Salmonella sp.	Fe₃O₄modified with streptavidin was used to capture biotinylated antibody and determine streptavidin interaction, reducing the transverse relaxation time of neighboring water molecules analyzed by NMR	10⁵CFU mL⁻¹	60	¹¹¹111 Zou, D.; Jin, L.; Wu, B.; Hu, L.; Chen, X.; Huang, G.; Zhang, J.; Int. Dairy J. 2019, 91, 82.
Peptide	Stachybotrys chartarum	peptide-labeled MNP fragments can be formed from Stachybotrys chartarum culture supernatant dripped onto a nano-wave; fragments can be split off and attracted by an external magnet, causing a golden coloration visible to the naked eye and indicating a positive reading	10-100 spores mL⁻¹	1	⁵³53 Suaifan, G. A.; Zourob, M.; Microchim. Acta 2019, 186, 230.
Antibodies (Abs) CSA-1-Ab	S. typhimurium	through the development of a nano platform, FPMNPs is possible to detect and capture S. typhimurium; the covalent binding of Abs facilitates the capture and immunomagnetic separation of the pathogen from a food matrix, allowing the obtaining of a SERS signal utilizing Raman	100 cells mL⁻¹	60	⁴¹41 Chattopadhyay, S.; Sabharwal, P. K.; Jain, S.; Kaur, A.; Singh, H.; Anal. Chim. Acta 2019, 1067, 98.
N-Succinyl feroxamine	Yersinia enterocolitica	by binding bacteria to the MNP conjugate due to the MNP surface’s electrostatic interactions and the feroxamine receptor, detection is possible	ND	60	¹¹²112 Martínez-Matamoros, D.; Castro-García, S.; Balado, M.; Matamoros-Veloza, A.; Camargo-Valero, M. A.; Cespedes, O.; Rodríguez, J.; Lemos, M. L.; Jiménez, C.; RSC Adv. 2019, 9, 13533.

Functionalized substances	Pathogens or analyte to detect	Operating principle	Limit of detection	Detection time / h	Reference
Thiol-ssDNA	Escherichia coli	a thiol-ssDNA-QD sensor was developed through a metal-thiol bond between the QD and the DNA system; bacteria in situ hybridization experiments were carried out, incubating cells with DNA; the cells were detected with an inverted fluorescence microscope	ND	1	¹¹⁴114 Wu, S. M.; Zhao, X.; Zhang, Z. L.; Xie, H. Y.; Tian, Z. Q.; Peng, J.; Lu, Z. X.; Pang, D. W.; Xie, Z. X.; ChemPhysChem 2006, 7, 1062.
Biotinylated bacteriophage	Escherichia coli	a specific bacteriophage for Escherichia coli was biotinylated in vivo, using a short peptide on the capsid surface marked with QDs coated with estraptivirin; phage infection allowed the visualization of the bacteria in water samples regardless of the presence of several mixtures of bacterial species	10 cells mL⁻¹	1	⁵⁷57 Edgar, R.; McKinstry, M.; Hwang, J.; Oppenheim, A. B.; Fekete, R. A.; Giulian, G.; Merril, C.; Nagashima, K.; Adhya, S.; Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 4841.
Antibody	Salmonella typhimurium, Escherichia coli O157:H7 and Listeria monocytogenes	the immuno-MNBs mixture allows to capture and magnetically separate three target bacteria in food samples; fluorescence intensity of MNB-cell-QD complexes determines the populations of pathogens	50 CFU mL⁻¹	2	¹¹⁵115 Wang, H.; Li, Y.; Wang, A.; Slavik, M.; J. Food Prot. 2011, 74, 2039.
Antibody IgG	Salmonella typhimurium	CdSe/ZnS QDs were immobilized onto a porous polycarbonate membrane having bound IgG antibodies specific for Salmonella tiphymurium; these nanostructures were used as fluorochromes during immunodetection; the capture of the cells was detected by confocal laser microscopy	100 cells mL⁻¹	2	¹¹⁶116 Jackeray, R.; Zainul Abid, C. K. V.; Singh, G.; Jain, S.; Chattopadhyaya, S.; Sapra, S.; Shrivastav, T. G.; Singh, H.; Talanta 2011, 84, 952.
Amino-modified aptamers	Salmonella typhimurium	the fluorescence detection of Salmonella typhimurium followed the incubation time and the addition of CD-aptamers; a linear relationship between the concentration of Salmonella typhimurium and the intensity of fluorescence was achieved	50 CFU mL⁻¹	ND	¹¹⁷117 Wang, R.; Xu, Y.; Zhang, T.; Jiang, Y.; Anal. Methods 2015, 7, 1701.
Antibody	Staphylococcus aureus	QDs coated with streptavidin and conjugated with biotin-labeled anti-S. aureus antibodies were developed as fluorescence markers; the fluorescence intensity of the bead-cell-QD complexes in the presence of S. aureus at 620 nm allows detection	10³CFU mL⁻¹	2	⁷⁹79 Yaohua, H.; Chengcheng, W.; Bing, B.; Mintong, L.; Wang, R.; Li, Y.; Int. J. Agric. Biol. Eng. 2014, 7, 77.
Antibody	Salmonella enteritidis	the specific interaction between Salmonella enteritidis and CdTe QDs modified with ligands conjugated to anti-Salmonella antibodies allows the capture and detection of Salmonella enteritidis by fluorescence microscopy	10²CFU mL⁻¹	2	⁷¹71 Wang, B.; Huang, X.; Ma, M.; Shi, Q.; Cai, Z.; Food Control. 2014, 35, 26.
Metal-organic frameworks	Escherichia coli O157:H7	CdS@ZIF-8 NPs as signal tags allow detection of Escherichia coli O157:H7; detection is made possible by differential pulse voltammetry when analyzing cadmium(II) ions released from CdS@ZIF-8 tags by HCl leaching	3 CFU mL⁻¹	1	⁸¹81 Zhong, M.; Yang, L.; Yang, H.; Cheng, C.; Deng, W.; Tan, Y.; Xie, Q.; Yao, S.; Biosens. Bioelectron. 2019, 126, 493.
Metronidazole (MET)	Porphyromonas gingivalis	the ability of oral epithelial cells (H413) with cCQDs to attack intracellular pathogens was studied; cell nuclei stained with DAPI produced a strong emission; it was found a cCQDs penetration capacity of 80% (m/m) using UV-Vis spectroscopy	0.26 µmol L⁻¹	180	⁵⁰50 Ardekani, S. M.; Dehghani, A.; Ye, P.; Nguyen, K.-A.; Gomes, V. G.; J. Colloid Interface Sci. 2019, 552, 378.
ZnO nanowire array (NWs)	Escherichia coli O157:H7	an aptamer as a recognition element made up of aligned ZnO NWs, decorated with AuNP and CdS QDs, allowed to form a nanoarray that can act as a self-amplified PEC biosensor	1.125 CFU mL⁻¹	60	⁴²42 Dong, X.; Shi, Z.; Xu, C.; Yang, C.; Chen, F.; Lei, M.; Wang, J.; Cui, Q.; Biosens. Bioelectron. 2020, 149, 111843.
Antibody	Salmonella sp.	considering that metal ion signals can be correlated with the number of bacterial cells captured by the sensor, QDs (CdTe) were studied by voltammetry after their acid dissolution using carbon electrodes screen-printed with bismuth(III) film formed in situ	4 CFU mL⁻¹	150	⁵⁵55 Murasova, P.; Kovarova, A.; Kasparova, J.; Brozkova, I.; Hamiot, A.; Pekarkova, J.; Dupuy, B.; Drbohlavova, J.; Bilkova, Z.; Korecka, L.; J. Electroanal. Chem. 2020, 114051.
Manganese dioxide nanoflowers (MnO₂NFs)	Escherichia coli O157:H7 Salmonella	target bacteria were conjugated to pAb-QDs@MnO₂nanocomposites, where antibodies (pAbs) help form MNB-bacteriaQM complexes, whose fluorescent intensity at the characteristic wavelength was measured to determine the presence of the target bacteria after separating QD and Mn²2 Bej, A. K.; Mahbubani, M. H.; PCR Methods Appl. 1992, 1, 151.+ ions	15, 40 CFU mL⁻¹	120	⁸⁰80 Xue, L.; Huang, F.; Hao, L.; Cai, G.; Zheng, L.; Li, Y.; Lin, J.; Food Chem. 2020, 126719.

Functionalized substances	Pathogens	Operating principle	Limit of detection	Detection time / h	Reference
Bovine serum albumin (BSA)	Escherichia coli O157:H7	immuno-SWNT, made up of goat anti-Escherichia coli O157 antibody conjugated to SWNT-BSA, makes it possible to recognize pathogenic Escherichia coli O157:H7 cells through specific antibody-antigen interactions	ND	1	¹¹⁸118 Elkin, T.; Jiang, X.; Taylor, S.; Lin, Y.; Gu, L.; Yang, H.; Brown, J.; Collins, S.; Sun, Y. P.; ChemBioChem 2005, 6, 640.
Galactose (Gal)	Escherichia coli O157:H7	SWNT, as a vehicle for multiple carbohydrate ligands, allows capturing pathogenic Escherichia coli in physiological solutions; furthermore, water-soluble SWNTs conjugated with galactose may exhibit inhibition of specific cellular responses	ND	1	¹¹⁹119 Gu, L.; Elkin, T.; Jiang, X.; Li, H.; Lin, Y.; Qu, L.; Tzeng, T.-R. J.; Joseph, R.; Sun, Y.-P.; Chem. Commun. 2005, 874.
Antibody (Ab)	Salmonella infantis	using the transduction capacity of CNTFETs combined with the antigen-antibody interaction recognition capacity produces a fast, sensitive, and tag-free biosensor for the selective detection of S. infantis	100 CFU mL⁻¹	1	⁴⁶46 Villamizar, R. A.; Maroto, A.; Rius, F. X.; Inza, I.; Figueras, M. J.; Biosens. Bioelectron. 2008, 24, 279.
Antibody (Ab)	Candida albicans	monoclonal anti-Candida antibodies adsorbed on SWCNT provide specific binding sites for fungal antigens; the methodology uses a FET in which SWCNTs form the conducting channel	50 CFU mL⁻¹	1	⁶³63 Villamizar, R. A.; Maroto, A.; Rius, F. X.; Sens. Actuators, B 2009, 136, 451.
Antibody (Ab)	Escherichia coli O157:H7 Bacteriophage T7	specific Ab-functionalized SWNTs-bonded gold electrodes act as chemoresistive biosensors; the functionalization with specific Ab of various microorganisms was carried out covalently	10⁵, 10³ CFU mL⁻¹	0.1	⁶⁴64 García-Aljaro, C.; Cella, L. N.; Shirale, D. J.; Park, M.; Muñoz, F. J.; Yates, M. V.; Mulchandani, A.; Biosens. Bioelectron. 2010, 26, 1437.
Antibody (Ab)	Salmonella enterica serovar Typhimurium	a bioconjugate of Ab/SWCNTs/HRP was used in ELISA experiments for high protein recovery; in this, the SWCNTs serve for the co-immobilization of antibodies and horseradish peroxidase	10³-10⁴CFU mL⁻¹	2	⁷⁶76 Chunglok, W.; Wuragil, D. K.; Oaew, S.; Somasundrum, M.; Surareungchai, W.; Biosens. Bioelectron. 2011, 26, 3584.
Antibody (Ab)	rotavirus	a graphene film with a uniform ripple structure on the surface shows high conductivity and rapid electron transfer properties, allowing it to work as an electrode in cyclic voltammetry	10⁵PFU mL⁻¹	ND	⁸⁷87 Liu, F.; Choi, K. S.; Park, T. J.; Lee, S. Y.; Seo, T. S.; BioChip J. 2011, 5, 123.
DNA	Escherichia coli O157:H7	the interaction between a DNA sequence and the MWCNTs during the preparation of a DNA sensor was investigated using Raman spectroscopy and Fourier transform infrared spectroscopy	1 nM	2	⁶⁶66 Thuy, N. T.; Tam, P. D.; Tuan, M. A.; Le, A.-T.; Van Thu, V.; Van Hieu, N.; Chien, N. D.; Curr. Appl. Phys. 2012, 12, 1553.
Pyrenil and aminemodified aptamers	Staphylococcus aureus	a network of SWCNTs acting as an ion-to-electron potentiometric transducer and anti-S. aureus aptamers, as the recognition element, showed a linear behavior between the potential and the bacteria concentration	800 CFU mL⁻¹	48	⁷⁸78 Zelada-Guillén, G. A.; Sebastián-Avila, J. L.; Blondeau, P.; Riu, J.; Rius, F. X.; Biosens. Bioelectron. 2012, 31, 226.
DNAzyme-labeled aptamer	Salmonella paratyphi A	aptamers with high binding capacity and specificity against Salmonella paratyphi A were developed; the aptamer-pathogen interaction was followed by fluorescence	10³CFU mL⁻¹	15	¹²⁰120 Yang, M.; Peng, Z.; Ning, Y.; Chen, Y.; Zhou, Q.; Deng, L.; Sensors 2013, 13, 6865.
Metal nanoparticle	Escherichia coli O157:H7	AuNPs-3D graphene interdigitated array electrodes were converted from polyimide film coated with the corresponding metal precursor-chitosan hydrogel ink and used to fabricate a flexible impedimetric immunosensor	100 CFU mL⁻¹	0.5	⁷⁰70 You, Z.; Qiu, Q.; Chen, H.; Feng, Y.; Wang, X.; Wang, Y.; Ying, Y.; Biosens. Bioelectron. 2020, 150, 111896.
Antibody	Yersinia enterocolitica	a biosensor based on SWCNT immobilized with anti-Yersinia antibody showed that, by linear scanning voltammetry, it is possible to detect the pathogen	10⁴CFU mL⁻¹	0.5	⁷³73 Sobhan, A.; Lee, J.; Park, M.-K.; Oh, J.-H.; LWT 2019, 108, 48.

Functionalized substances	Pathogens or analyte to detect	Operating principle	Limit of detection	Detection time / h	Reference
Terbium-metal organic framework Tb(BTC) (H₂O)₆(Tb-MOF)	Bacillus anthracis (dipicolinic acid DPA biomarker)	by mixing the fluorescence probe Tb-MOF with DPA, a rapid reduction in the photoluminescence signal can be obtained, maintaining a highly linear relationship with increasing DPA concentrations	0.04 nmol L⁻¹	0.2	¹²⁴124 Bhardwaj, N.; Bhardwaj, S.; Mehta, J.; Kim, K.-H.; Deep, A.; Biosens. Bioelectron. 2016, 86, 799.
NH₂-MIL-53(Fe)	Staphylococcus aureus	the use of glutaraldehyde as a crosslinking agent allows the conjugation of a MOF with bacteriophages, obtaining a biosensor that detects S. aureus through photoluminescence extinction	31 CFU mL⁻¹	0.3	¹²³123 Bhardwaj, N.; Bhardwaj, S. K.; Mehta, J.; Kim, K.-H.; Deep, A.; ACS Appl. Mater. Interfaces 2017, 9, 33589.
Copper metal-organic framework nanoparticles (Cu-MOF-NPs)	Escherichia coli	Cu-MOF-NP improved biological activity by increasing the dose of particles against various pathogens (five bacteria strains, Gram-positive and Gram-negative) compared to an antibacterial agent	50 µg mL⁻¹	ND	¹²⁵125 Sheta, S. M.; El-Sheikh, S. M.; Abd-Elzaher, M. M.; Dalton Trans. 2018, 47, 4847.
JXNU-4 [(Me₂NH₂)₂(Zn₆(µ₄-O)(ad)₄(BPDC)₄]_n	Bacillus anthracis (dipicolinic acid DPA biomarker)	a dual emission hybrid MOF (Tb³⁺@MOF) allowed radiometric detection of DPA; the formation of the Tb-DPA complex produces a sensitization of the antenna, increasing the emission of Tb³⁺	3.6 nmol L⁻¹	0.2	⁴⁹49 Zhang, D.; Zhou, Y.; Cuan, J.; Gan, N.; CrystEngComm 2018, 20, 1264.
Polyaniline (PANI)	Escherichia coli O157:H7	the monitoring and quantification of the interaction between an aptamer and E. coli using methylene blue as an electrochemical indicator were carried out by differential pulse voltammetry; the MB-E. coli O157:H7 interaction generates a signal proportional to the concentration of E. coli O157:H7	2 CFU mL⁻¹	0.3	⁷²72 Shahrokhian, S.; Ranjbar, S.; Analyst 2018, 143, 3191.
Copper(II) tetrakis (4-carboxyphenyl)porphyrin nanosheets (MOF-NSs)	Salmonella enterica, Listeria monocytogenes, and Vibrio parahemolyticus	the MOF-NSs possess a distinguished affinity for ssDNA, which causes the labeled DNA’s fluorescence to be quenched; the labeled duplex is released on binding to the target pathogen DNA, and the label’s fluorescence is restored	28, 35 and 15 pmol L⁻¹	1	⁶⁹69 Qiu, Q.; Chen, H.; Ying, S.; Sharif, S.; You, Z.; Wang, Y.; Ying, Y.; Microchim. Acta 2019, 186, 93.
Aptamer and DNA	Pseudomonas aeruginosa	decomposition of H₂O₂catalyzed by a Cu-ZrMOF@Aptamer@DNA nanocomposite acted as a signal probe showing conductivity changes due to electron transfer during successful detection	2 CFU mL⁻¹	2	⁸⁵85 Zhang, X.; Xie, G.; Gou, D.; Luo, P.; Yao, Y.; Chen, H.; Biosens. Bioelectron. 2019, 142, 111486.
Antibodies	Staphylococcus aureus	an antibody-conjugated bioprobe (Ab/NH₂-MIL-53) incubated with varying concentrations of Staphylococcus aureus maintained a strong inverse correlation in which the decrease in fluorescence intensity was accompanied by an increase in bacterial count	85 CFU mL⁻¹	ND	¹²⁶126 Bhardwaj, N.; Bhardwaj, S. K.; Bhatt, D.; Tuteja, S. K.; Kim, K.-H.; Deep, A.; Anal. Methods 2019, 11, 917.
Polyaniline (PANI)	Escherichia coli	a biosensor electrode was developed by conjugating anti-E. coli antibodies and Cu₃(BTC)₂-PANI on an indium-tin-oxide substrate; this biosensor allowed detection of very low concentrations of E. coli using electrochemical impedance spectroscopy	2 CFU mL⁻¹	2	⁸⁸88 Gupta, A.; Bhardwaj, S. K.; Sharma, A. L.; Kim, K.-H.; Deep, A.; Environ. Res. 2019, 171, 395.

[1] *e-mail: casierraa@unal.edu.co

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Nanobiosensors for Pathogenic Agents Detection