pubs.acs.org/ac Article Bacteria Detection at a Single-Cell Level through a Cyanotype-Based Photochemical Reaction Jiri Dietvorst, Amparo Ferrer-Vilanova, Sharath Narayana Iyengar, Aman Russom, Núria Vigués, Jordi Mas, Lluïsa Vilaplana, Maria-Pilar Marco, Gonzalo Guirado,* and Xavier Muñoz-Berbel* Cite This: Anal. Chem. 2022, 94, 787−792 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles. ABSTRACT: The detection of living organisms at very low concentrations is necessary for the early diagnosis of bacterial infections, but it is still challenging as there is a need for signal Downloaded via 83.41.213.147 on February 3, 2022 at 14:32:54 (UTC). amplification. Cell culture, nucleic acid amplification, or nano- structure-based signal enhancement are the most common amplification methods, relying on long, tedious, complex, or expensive procedures. Here, we present a cyanotype-based photochemical amplification reaction enabling the detection of low bacterial concentrations up to a single-cell level. Photocatalysis is induced with visible light and requires bacterial metabolism of iron-based compounds to produce Prussian Blue. Bacterial activity is thus detected through the formation of an observable blue precipitate within 3 h of the reaction, which corresponds to the concentration of living organisms. The short time-to-result and simplicity of the reaction are expected to strongly impact the clinical diagnosis of infectious diseases. ■ INTRODUCTION Bacteria are microorganisms related to important healthcare increase its sensitivity up to a single bacterium level.10 However, the long time-to-result of pre-enrichment methods and safety problems including infectious diseases,1 food (between 24 and 36 h), the cost, and complexity of SERS and poisoning,2 and water pollution.3 Lower respiratory infections PCR11 limit their use in the early diagnosis of bacterial and diarrheal diseases are now positioned as the fourth and infections. Conversely, photochemical reactions initiated and catalyzed eighth cause of death worldwide.4 Diarrheal diseases, resulting by light are good candidates for bacterial detection for being from the consumption of contaminated food and water, are fast, simple, and cheap. Among them, a cyanotype reaction is estimated to cause 550 million foodborne illnesses and 230 000 considered in the first place since employing iron-based deaths every year, where Escherichia coli is one of the most complexes is susceptible to react with bacterial components, common foodborne pathogens.5 To minimize the impact of i.e., ferricyanide and ammonium ferric citrate. In cyanotype, bacterial infections, these microorganisms should be detected UV light is used to photoactivate ammonium ferric citrate, at an early stage of infection, when only a few bacteria are which reacts with ferricyanide to produce Prussian Blue (PB) present. This is particularly important in the case of sepsis, particles and an intense blue color.12 The mechanism of the requiring the detection of less than 100 colony forming units reaction entails two main steps (Figure 1, left): (i) the (CFU)/mL in the bloodstream,6 since every hour delay in the photochemical dissociation of iron citrate complexes, involving detection of bacteria increases patients’ mortality by 10%.7 the reduction of iron(III) to iron(II) and the concomitant Current gold standard methods for microbial detection oxidation of citrate;13 and (ii) the reaction of free iron(II) ions involve amplification steps to reach the appropriate bacteria with ferricyanide to produce PB. It is widely accepted that detectability. The three most common amplification methods citrate oxidation starts with the formation of a highly reactive are: (i) pre-enrichment by cell culture, where bacterial citrate radical intermediate. Since poorly stable, citrate radicals proliferation increases cell population up to a detectable magnitude;8 (ii) nucleic acid amplification, such as the polymerase chain reaction (PCR),9 where a specific target Received: August 5, 2021 sequence is copied many times in repeated cycles until Accepted: December 6, 2021 reaching a detectable number of copies; and (iii) nanostruc- Published: December 21, 2021 ture-based signal enhancement, e.g., surface-enhanced Raman spectroscopy (SERS), where a Raman scattering signal is amplified by the use of nanoscale roughness features that © 2021 The Authors. Published by American Chemical Society https://doi.org/10.1021/acs.analchem.1c03326 787 Anal. Chem. 2022, 94, 787−792 Analytical Chemistry pubs.acs.org/ac Article Figure 1. Illustration of the mechanism for the classic cyanotype (left) and the proposed cyanotype-based reaction for bacterial detection (right). In both cases, the mechanism is divided into two reactions, namely, (i) photodissociation of iron citrate and (ii) free iron ions’ reaction with hexacyanoferrate species. Below, the main components of the electron transport chain are presented with the corresponding redox potential and role in the reaction. dissociate rapidly to other radicals and reactive oxygen species, Preparation of McFarland Standard and Bacterial which react with other iron citrate molecules, accelerating their Samples. The McFarland standards are employed to dissociation and promoting PB formation.14 This photo- standardize antimicrobial susceptibility tests. The optical catalytic and radical mechanism makes the reaction very fast density of these standards is used as a reference to adjust the and promising for biosensing, but the need for UV radiation to turbidity of bacterial suspensions used in the assay. In this case, initiate the reaction, which is toxic to bacteria, limits its bacterial colonies were inoculated into a sterile saline solution application to bacterial detection. (0.85% NaCl w/v in water) using a cotton swab to obtain an In this work, a cyanotype reaction is modified in a way that optical density of 0.1 au at 600 nm, which corresponds to the UV light is substituted by less energetic visible light and microbiology standard McFarland standard no. 0.5. The coupled to bacterial metabolism for sensitive and selective bacterial culture was prepared to match the same optical detection of live bacteria. density, 0.160 for this device, which corresponded to 108 ■ MATERIALS AND METHODS Reagents. The precursor reagents for the cyanotype-based CFU/mL of bacteria. To adjust bacterial counts to the experiment of interest, the 0.5 McFarland solution was diluted in an MH medium. The optical density of the 0.5 McFarland reaction were composed of ammonium ferric citrate (Sigma standard was determined using a spectrophotometer (Smart- Aldrich) and potassium hexacyanoferrate (Sigma Aldrich). A spec Plus spectrophotometer, Bio-Rad, California). precursor solution was prepared in Mueller Hinton (MH) Cyanotype-Based Assays for Bacterial Detection. The media (Sigma Aldrich) and adjusted to an acidic pH of 6.5 to assays for bacterial detection using the cyanotype reaction were avoid PB decomposition. based on the standard broth microdilution protocol defined by Bacterial Culture. E. coli (ATCC 25922) was donated by the European Committee on Antimicrobial Susceptibility Prof. Herman Goossens from the University of Antwerp. Testing (EUCAST), with the small variations detailed below Bacteria were grown overnight on an LB agar plate at 37 °C, resulting from the photocatalytic nature of the detection plated using the standard streaking method. These streaks were reaction. The assays were performed in 96-well plates (Cell taken from plated culture, stored at 4 °C in the fridge for a culture microplate 96 well, PS, U-bottom, Cellstar), with the maximum of 2 weeks. optical measurements carried out using a microplate reader 788 https://doi.org/10.1021/acs.analchem.1c03326 Anal. Chem. 2022, 94, 787−792 Analytical Chemistry pubs.acs.org/ac Article Figure 2. Proof of concept of the cyanotype-based reaction. Variation of the absorbance magnitude at 720 nm, corresponding to the PB peak, for samples containing the culture medium alone, cyanotype precursors alone, bacteria alone, or precursors and bacteria, either in the dark (a) or under continuous irradiation. (b) Inset, images of the aspect of the reaction Eppendorf tubes, with a clear blue precipitate in the case of light irradiation. The visible absorbance spectra of the previous light samples after 7 h of incubation are illustrated in (c), where the peaks corresponding to bacterial scattering, iron absorption, and the absorption/scattering of PB molecules are identified. In (d), the response of the cyanotype-based reaction with two hexacyanoferrate species, i.e., ferricyanide and ferrocyanide, is compared and is found to have similar absorbance values. Experimental conditions: a bacterial starting concentration of 106 CFU/mL of E. coli ATCC 25922 was used. The reagents refer to a 1.25 mM ferricyanide and 5 mM ferric ammonium citrate concentration (n = 2). (Thermo-Fisher) in the range between 400 and 800 nm (step follows a typical proliferation curve for E. coli. Since both size of 10 nm) and with measurements being performed every bacterial samples with and without cyanotype precursors show hour. The microtiter plate was kept at 37 °C inside an similar growth curves, it is evident that these reagents do not incubator. Bacterial culture and reagents were transferred to compromise bacterial growth at the experimental conditions the 96-well plate, for a total of 200 μL, composed of 100 μL of under study. bacteria, 50 μL of MH, and 25 μL of each precursor to adjust In light conditions (Figure 2b), ultraviolet (UV) radiation the final concentration to that required in each experiment. used in classical cyanotype is substituted by less energetic The microtiter plate was kept at 37 °C inside an incubator, visible light, which is not sufficient to directly catalyze the which contained a white light-emitting diode (LED) lamp photochemical dissociation of iron citrate necessary for the (Matel 6400 K, 15 W 1500 lumen) to enable homogeneous production of PB (orange curve in the figure). PB formation is and constant illumination to the sample from above. The only evident when both cyanotype precursors and bacteria are sample was kept inside the illumination chamber in-between present and continuously irradiated. PB appears as a blue measurements. The total illumination time was 5 h, after which precipitate (image inset the figure) and results in a large the plate was kept inside the heated microplate reader for the increase in the absorbance magnitude at 720 nm, which is sequential automatic measurements every hour. associated with the sum of the scattering and absorption of PB ■ RESULTS AND DISCUSSION Bacterial metabolism is known to reduce iron-based molecules particles (Figure 2c). When analyzing the contribution of each reaction component to the formation of PB, it is evident that both iron citrate and hexacyanoferrate molecules are necessary such as ferricyanide,15−17 Presto Blue,18 or even PB19 by a (Supporting Information, S1). However, the reaction mecha- reaction with components of the electron transport chain nism does not depend on the oxidative state of the (ETC). Iron citrate is also selectively recognized and used by hexacyanoferrate ions and similar results are obtained when microorganisms as an iron source,20 where citrate has a dual substituting ferricyanide by its reduced form ferrocyanide role, as an iron chelator for transport of iron ions into the cell21 (Figure 2d). Considering that the formation of PB requires the and as a key intermediate in the citric acid cycle.22 However, presence of free iron ions with a specific oxidative state to react the bacterial metabolism of iron citrate alone does not release with hexacyanoferrate complexes, i.e., iron(II) in the case of enough free iron ions to mediate the formation of PB directly. ferricyanide and iron(III) for ferrocyanide, this result suggests Without photoactivation of iron citrate (i.e., dark conditions that both iron(II) and iron(III) ions are produced after shown in Figure 2a), the spectroscopic analysis of PB at 720 photometabolic dissociation of iron citrate molecules. How- nm (corresponding to its absorption peak) does not show PB ever, at this stage, it has not been possible to confirm the formation either in the presence or the absence of bacteria. presence of free iron(III) (Supporting Information, S2). The increase in absorbance reported in the dark samples Iron(III) detection is based on the specific reaction between containing bacteria is associated with cell scattering and acetylsalicylic acid and free iron(III) ions, resulting in the 789 https://doi.org/10.1021/acs.analchem.1c03326 Anal. Chem. 2022, 94, 787−792 Analytical Chemistry pubs.acs.org/ac Article formation of a complex with an intense purple color and absorbance at 565 nm. Complex formation has not been observed in any of the previous reaction conditions. The reason for this may be a fast reaction kinetic between free iron(III) and hexacyanoferrate ions to form PB and thus, a short lifetime of free iron ions. In addition, the lack of a reaction between ferrocyanide and iron citrate in light conditions (Figure 2d, orange line in the ferrocyanide plot), which should produce PB directly through the classical cyanotype reaction, confirms the slow kinetics of the photochemical dissociation of iron citrate molecules with visible light. Considering previous results, the reaction mechanism presented in Figure 1 right is proposed. It involves the diffusion of iron citrate into the periplasmic region of bacteria, where it is excited to an intermediate state [Fe(III)-Cit]° by proteins and mediators of the bacterial ETC, e.g., cytochromes and ubiquinone. Thus, the intermediate formation requires intact bacterial membranes, so the presence of viable bacteria starts the reaction. This excited intermediate is susceptible to photochemical dissociation by visible light, releasing iron(II) ions and other radical intermediates, resulting from the photo- oxidation of citrate to the medium. Once dissociated, a photocatalytic cascade begins, where radicals react with other iron citrate molecules inducing their dissociation. Part of the iron(II) ions are reoxidized to iron(III) by the effect of the radicals and hydrogen peroxide produced as side products by the reaction. Bacterial metabolism additionally reduces intermediate iron compounds, metabolizes citrate, and reduces ferricyanide to ferrocyanide. Therefore, independently of the Figure 3. Optimization of the composition of the cyanotype precursor solution. (a) Image illustrating the color of the reaction solution after initial composition of the cyanotype precursor solution, the 5 h of incubation with several precursor solutions dilutions and photometabolic activation of the reagents results in a mixture reagents proportions. The absorbance magnitude of the most of free iron(II) and iron(III) ions, ferricyanide and representative samples is illustrated in (b). Red arrows indicate the ferrocyanide, resulting in the fast formation of PB molecules. difference between the control sample without bacteria and the This photometabolic activation only takes place when using sample containing 5 × 105 CFU/mL E. coli ATCC 25922 for the diluted cyanotype precursor solutions containing iron citrate concentration and proportions considered optimal (n = 3). concentrations below 10 mM and maintaining a molar ratio citrate:hexacyanoferrate in the range between 4 and 8 (Figure responsible to produce Cit°, other radicals, and reactive 3a; above 10 mM iron citrate, a classical cyanotype reaction oxygen species in a cascade reaction, resulting in the occurs, where PB is formed spontaneously without the need production of free iron ions and PB molecules. This reaction for bacteria). From all combinations under study, the takes place in 3 h in samples’ containing bacterial proportion 2.5:0.62 mM iron citrate:hexacyanoferrate presents concentrations as low as 100 CFU/mL, which correspond to the highest signal-to-noise ratio for being the one with the an average of 10 CFUs for 100 μL of samples. The assay, lowest background noise, which corresponds to the signal of however, reaches single bacterium sensitivity and is highly the control samples without bacteria (Figure 3b; the repetitive, as shown in Figure 4c. In the figure, samples are absorbance magnitude and spectra of all conditions are grouped according to the initial bacterial concentration in (i) presented in the Supporting Information, S3). low bacteria (between 1 and 2 CFUs), (ii) middle (between 5 When the photometabolic reaction is conducted at different and 8 CFUs), and (iii) high (above 10 CFUs). It is clear from bacterial concentrations (Figure 4b), high signal amplification the results that one to two bacterial cells are not enough to is obtained in all samples. A particular behavior is observed in induce bacterial proliferation since the second absorbance samples below 105 CFU/mL, where bacterial activity is slower increase associated with bacterial scattering is absent in this than photocatalysis. In this case, a sudden increase in case. Control samples without bacteria or containing dead absorbance resulting from PB formation is reported within 3 bacteria do not present the initial sudden increase, confirming h of the reaction, followed by stabilization and a second that this is a photometabolic process only happening when live increase attributed to bacterial scattering. bacteria are present at the initial stages of the reaction (even if This fast PB formation is independent of the bacterial they die afterward). However, the control without bacteria is concentration for samples below 105 CFU/mL and selective not completely flat but presents some increase that may be due since it only happens in light samples containing bacteria and to some late reaction between precursor components (after 11 not in the dark ones (Figure 4a) or controls without bacteria. h) to produce PB (e.g., direct cyanotype reaction). The Therefore, the photocatalytic reaction is much faster than cyanotype-based reaction has no cross-reactivity with compo- bacterial metabolism, which is in agreement with the nents present in the serum, and blood (Supporting mechanism proposed in Figure 1 right. That is, bacteria Information, S4) since there is no PB formation in those initiate the reaction but, after that, the light becomes serum/blood samples that do not contain bacteria. However, 790 https://doi.org/10.1021/acs.analchem.1c03326 Anal. Chem. 2022, 94, 787−792 Analytical Chemistry pubs.acs.org/ac Article Figure 4. Evaluation of the response of the cyanotype-based reaction with different bacterial concentrations. Variation of the absorbance magnitude at 720 nm for samples containing cyanotype precursor solutions and bacteria at concentrations between 100 and 108 CFU/mL in the dark (a) or after 5 h of continuous irradiation (b) (n = 3). In (c), the replicas of the 100 CFU/mL sample are represented individually to evaluate the variability in the single bacterium detection and compared to controls without bacteria and with dead bacteria. the reaction in these complex matrices requires the Additional experiments for the evaluation of the role of optimization of the composition of the precursor solution to cyanotype precursors in the reaction (S1); additional be able to detect PB formation within 3 h (now requiring 5 h experiments for the evaluation of the presence of free for 102 CFU/mL samples). It is important to remark that the iron(III) molecules during the cyanotype-based reaction photochemical reaction has been demonstrated for E. coli, (S2); additional experiments to study the proportion which is used as a model microorganism. However, proteins and concentration of the precursor solution components and redox mediators present in the ETC are very conservative (S3); additional experiments to evaluate the cross- between bacterial species, and such a reaction should be reactivity of the cyanotype-based reagent with serum and applicable to a wide number of Gram-positive and Gram- blood components (S4) (PDF) ■ negative bacteria. In this sense, the reaction has been already demonstrated by the Gram-positive microorganism Staph- ylococcus aureus in a recent article of the group focused on AUTHOR INFORMATION sepsis diagnostics.23 Corresponding Authors ■ CONCLUSIONS In summary, the substitution of UV by visible light in a Gonzalo Guirado − Departament de Química, Universitat Autònoma de Barcelona, Bellaterra (Barcelona) 08193, Spain; orcid.org/0000-0003-2128-7007; cyanotype photochemical reaction enables the coupling of PB Email: Gonzalo.Guirado@uab.cat formation to bacterial metabolism. The reaction mechanism Xavier Muñoz-Berbel − Instituto de Microelectrónica de involves the photoactivation of iron citrate molecules into an Barcelona (IMB-CNM, CSIC), Bellaterra (Barcelona) excited intermediate state by reactive components in the 08193, Spain; orcid.org/0000-0002-6447-5756; electron transport chain, which is susceptible to photo- Email: xavier.munoz@imb-cnm.csic.es activation with visible light to produce free iron ions and Authors citrate radicals. Iron ions react with hexacyanoferrate molecules Jiri Dietvorst − Instituto de Microelectrónica de Barcelona to produce PB particles in less than 3 h, a process catalyzed by (IMB-CNM, CSIC), Bellaterra (Barcelona) 08193, Spain; citrate radicals that initiate a cascade reaction with other iron Nanobiotechnology for diagnostics (Nb4D), Department of citrate molecules to produce more radicals and free iron ions. Chemical and Biomolecular Nanotechnology, Institute for The reaction is very selective, requiring the presence of light Advanced Chemistry of Catalonia (IQAC, CSIC), Barcelona and bacteria, and sensitive since it may be activated by a single 08034, Spain microorganism. Components of complex biological fluids such Amparo Ferrer-Vilanova − Instituto de Microelectrónica de as serum or blood do not present cross-reactivity with the Barcelona (IMB-CNM, CSIC), Bellaterra (Barcelona) reaction precursors, making it possible to detect very low 08193, Spain; Departament de Química, Universitat bacterial concentrations in a short time and without sample Autònoma de Barcelona, Bellaterra (Barcelona) 08193, pretreatment. The simplicity, selectivity, and short time-to- Spain; orcid.org/0000-0002-4223-8442 result of this photometabolic reaction are envisioned to have Sharath Narayana Iyengar − Division of Nanobiotechnology, an impact on clinical diagnosis of bacterial infections, in Department of Protein Science, Science for life laboratory, particular in sepsis diagnostics where early detection of bacteria KTH Royal Institute of Technology, Stockholm 17165, in the bloodstream is fundamental to improve the prognosis of Sweden; orcid.org/0000-0001-5348-3526 the pathology. This protocol may be also used for fast Aman Russom − Division of Nanobiotechnology, Department antibiotic susceptibility testing, the determination of minimal of Protein Science, Science for life laboratory, KTH Royal inhibitory concentration, and the identification of resistant Institute of Technology, Stockholm 17165, Sweden bacteria. Núria Vigués − Departament of Genetics and Microbiology, ■ * ASSOCIATED CONTENT sı Supporting Information Universitat Autònoma de Barcelona, Bellaterra (Barcelona) 08193, Spain Jordi Mas − Departament of Genetics and Microbiology, The Supporting Information is available free of charge at Universitat Autònoma de Barcelona, Bellaterra (Barcelona) https://pubs.acs.org/doi/10.1021/acs.analchem.1c03326. 08193, Spain 791 https://doi.org/10.1021/acs.analchem.1c03326 Anal. Chem. 2022, 94, 787−792 Analytical Chemistry pubs.acs.org/ac Article Lluïsa Vilaplana − Nanobiotechnology for diagnostics (15) Pujol-Vila, F.; Vigués, N.; Guerrero-Navarro, A.; Jiménez, S.; (Nb4D), Department of Chemical and Biomolecular Gómez, D.; Fernández, M.; Bori, J.; Vallès, B.; Riva, M. C.; Muñoz- Nanotechnology, Institute for Advanced Chemistry of Berbel, X.; Mas, J. Anal. Chim. Acta 2016, 910, 60−67. Catalonia (IQAC, CSIC), Barcelona 08034, Spain; CIBER (16) Pujol-Vila, F.; Giménez-Gómez, P.; Santamaria, N.; Antúnez, B.; Vigués, N.; Díaz-González, M.; Jiménez-Jorquera, C.; Mas, J.; de Bioingeniería, Biomateriales y Nanomedicina (CIBER- Sacristán, J.; Muñoz-Berbel, X. Sens. Actuators, B 2016, 222, 55−62. BBN), Barcelona 08034, Spain (17) Pujol-Vila, F.; Dietvorst, J.; Gall-Mas, L.; Díaz-González, M.; Maria-Pilar Marco − Nanobiotechnology for diagnostics Vigués, N.; Mas, J.; Muñoz-Berbel, X. J. Colloid Interface Sci. 2018, (Nb4D), Department of Chemical and Biomolecular 511, 251−258. Nanotechnology, Institute for Advanced Chemistry of (18) Lall, N.; Henley-Smith, C. J.; De Canha, M. N.; Oosthuizen, C. Catalonia (IQAC, CSIC), Barcelona 08034, Spain; CIBER B.; Berrington, D. Int. J. Microbiol. 2013, 2013, 1−5. de Bioingeniería, Biomateriales y Nanomedicina (CIBER- (19) Ferrer-Vilanova, A.; Alonso, Y.; Dietvorst, J.; Pérez-Montero, BBN), Barcelona 08034, Spain; orcid.org/0000-0002- M.; Rodríguez-Rodríguez, R.; Ivanova, K.; Tzanov, T.; Vigués, N.; 4064-1668 Mas, J.; Guirado, G.; Muñoz-Berbel, X. Ultrason. Sonochem. 2021, 70, No. 105317. Complete contact information is available at: (20) Wagegg, W.; Braun, V. J. Bacteriol. 1981, 145, 156−163. https://pubs.acs.org/10.1021/acs.analchem.1c03326 (21) Mahren, S.; Schnell, H.; Braun, V. Arch. Microbiol. 2005, 184, 175−186. Notes (22) Frawley, E. R.; Fang, F. C. The Ins and Outs of Bacterial Iron The authors declare no competing financial interest. Metabolism. In Molecular Microbiology; Blackwell Publishing Ltd., Aug ■ 1, 2014; pp 609−616. (23) Iyengar, S. N.; Dietvorst, J.; Ferrer-Vilanova, A.; Guirado, G.; ACKNOWLEDGMENTS Muñoz-Berbel, X.; Russom, A. ACS Sens. 2021, 6, 3357−3366. This project has received funding from the European Union Horizon 2020 Research and Innovation Programme under the Marie Skłodow-ska-Curie grant agreement no. 675412 as a part of the consortium New Diagnostics for Infectious Diseases (ND4ID). G.G. thanks the Ministerio de Ciencia e Innovación of Spain for financial support through the project PID2019- 106171RB-I00. Patent (P42106384SE00) based on part of this work was granted. M.-P.M. and L.V. would like to thank the Ministry of Science and Innovation (SAF2015-67476-R and RTI2018-096278-B-C21) and Fundación Marató de TV3 (TV32018-201825-30-31). ■ REFERENCES (1) Vouga, M.; Greub, G. Clin. Microbiol. Infect. 2016, 22, 12−21. (2) Abou Zeid, A. A.; El Sherbeeny, M. R. Zentralblatt Bakteriol. Parasitenkd. Infekt. Hyg. Zweite Abteilung 1975, 130, 314−333. (3) Cabral, J. P. S. Water Microbiology. Bacterial Pathogens and Water. In International Journal of Environmental Research and Public Health; Molecular Diversity Preservation International, Oct 15, 2010; pp 3657−3703. (4) The top 10 causes of death. https://www.who.int/news-room/ fact-sheets/detail/the-top-10-causes-of-death (accessed Jun 22, 2021). (5) Food safety. https://www.who.int/news-room/fact-sheets/ detail/food-safety (accessed Jun 22, 2021). (6) Yagupsky, P.; Nolte, F. S. Clin. Microbiol. Rev. 1990, 3, 269−279. (7) Kumar, A.; Roberts, D.; Wood, K. E.; Light, B.; Parrillo, J. E.; Sharma, S.; Suppes, R.; Feinstein, D.; Zanotti, S.; Taiberg, L.; Gurka, D.; Kumar, A.; Cheang, M. Crit. Care Med. 2006, 34, 1589−1596. (8) Roszak, D. B.; Colwell, R. R. Appl. Environ. Microbiol. 1987, 53, 2889−2893. (9) Brakstad, O. G.; Aasbakk, K.; Maeland, J. A. J. Clin. Microbiol. 1992, 30, 1654−1660. (10) Lin, H.-Y.; Huang, C.-H.; Hsieh, W.-H.; Liu, L.-H.; Lin, Y.-C.; Chu, C.-C.; Wang, S.-T.; Kuo, I.-T.; Chau, L.-K.; Yang, C.-Y. Small 2014, 10, 4700−4710. (11) Sinha, M.; Jupe, J.; Mack, H.; Coleman, T. P.; Lawrence, S. M.; Fraley, S. I. Emerging Technologies for Molecular Diagnosis of Sepsis. In Clinical Microbiology Reviews; American Society for Microbiology, April 1, 2018. (12) Lawrence, G. D.; Fishelson, S. J. Chem. Educ. 1999, 76, No. 1216A. (13) Feng, W.; Nansheng, D. Chemosphere 2000, 41, 1137−1147. (14) Seraghni, N.; Belattar, S.; Mameri, Y.; Debbache, N.; Sehili, T. Int. J. Photoenergy 2012, 2012, 1−10. 792 https://doi.org/10.1021/acs.analchem.1c03326 Anal. Chem. 2022, 94, 787−792