Bringing the interaction of silver nanoparticles with bacteria to light

Abstract

In past decades, the exploitation of silver nanoparticles in novel antibacterial and detection devices has risen to prominence, owing to the well-known specific interaction of silver with bacteria. The vast majority of the investigations focus on the investigation over the mechanism of action underpinning bacterial eradication, while few efforts have been devoted to the study of the modification of silver optical properties upon interaction with bacteria. Specifically, given the characteristic localized surface plasmon resonance of silver nanostructures, which is sensitive to changes in the charge carrier density or in the dielectric environment, these systems can offer a handle in the detection of bacteria pathogens. In this review, we present the state of art of the research activity on the interaction of silver nanoparticles with bacteria, with strong emphasis on the modification of their optical properties. This may indeed lead to easy color reading of bacterial tests and pave the way to the development of nanotechnologic silver-based bacterial detection systems and drug-screening platforms.

I. INTRODUCTION

One of the great advances in medical science was the discovery of antibiotics, which provided humankind with an effective tool to fight off bacterial infections. However, in recent years the extensive use of antibiotics has led to bacteria developing resistance to drug therapy and the emergence of multidrug-resistance (MDR) in mutant species, which poses an obvious threat to human welfare.^1–3 Hence the need arises for researching alternatives to antibiotics that will overcome the rise of drug-resistant microbes: in this context, the potential of metals, and in particular silver, as an option for antibacterial devices has been recognized. Historically, silver has been widely regarded for its antimicrobial properties.⁴ Although much remains to be understood about the mechanism that contributes to its bactericidal action, advancements in research have made it possible to study more closely the interaction between the metal and the pathogens in order to obtain a clearer picture of the underlying biochemical mechanism. In particular, recent advances in nanotechnology have piqued the interest of researchers into the interaction between silver nanoparticles (AgNPs) and bacteria. This can be exploited for useful applications in the medical field, for instance, in rendering metallic surgical implants safer by inhibiting pathogen growth,⁵ as well as in surface and device sanitization and in the design and fabrication of bio-detectors for environmental and food-related monitoring.^6,7 Many studies concerning AgNPs point out the relatively high surface-to-volume ratio of these nano-objects, which ensures a large effective area of interaction with the pathogens per unit mass. This in turn leads to larger inhibition of the bacteria virulence when using nanoparticles as opposed to a flat metal surface.

Although silver's bactericidal effect has been studied for several decades, the modulation of its optical properties upon interaction with bacteria has been largely overlooked. This is a pity, since investigations into these effects can permit more insight into the biophysics underpinning their interaction. In addition, the changes in optical properties of AgNPs when interacting with bacteria can indeed be used as a key parameter to monitor the presence of pathogens and to build up drug-screening platforms. Silver nanoparticles in general exhibit a well-defined optical resonance owing to the confinement of their surface plasmon, the so-called localized surface plasmon resonance (LSPR). Such a spectroscopic feature is extremely sensible to the dielectric function of the surrounding medium, to the free electron density of the NPs, and to the geometry of the object that ultimately dictates the degree of surface plasmon confinement.⁸ Since bacteria are able to modify all these above-mentioned parameters, i.e., by causing NPs' aggregation/structural modifications and/or by modifying the charge carrier density due to Ag oxidative dissolution, a careful evaluation of the LSPR position and intensity upon AgNPs' exposure to bacteria can, in principle, give precious insights into the interaction process.

Although several works have shown that the silver LSPR is strongly modified in biologically relevant environments and upon exposure to actual bacterial cells, this effect does not usually represent the main topic of study. Thus, the scope of this review is to extract and gather the most relevant works dealing with these aspects, with the view to bringing the interaction between AgNPs and bacteria to a new light. The study of the relationship between Ag optical features and bacteria can be of broad significance, since a variety of inorganic and organic nanostructured materials displaying bio-activity,⁹ including metals (i.e., gold and copper), metal oxides (i.e., TiO₂ and ZnO), and organic systems (i.e., graphene), also exhibit well-defined optical resonances that can undergo dramatic changes when in contact with bacteria. These peculiarities can be practically exploited to build up bacterial sensors and drug-screening platforms, for instance to evaluate the activity of bacteria against selected drugs and antibiotics.

In Sec. II, we will give a brief account on the main mechanism driving AgNP modification upon interaction with bacteria, which lies on its oxidative dissolution and release of Ag⁺ ions. In Sec. III, we first introduce the well-known optical properties of AgNPs resulting from the localized surface plasmon resonance. Then, we will present a few selected examples in which the LSPR undergoes changes in position, shape, and intensity when in contact with biological species and whole bacterial cells. In Sec. IV, we will briefly summarize those findings and give a perspective on the possible developments of such a promising research topic.

II. MECHANISM OF INTERACTION OF SILVER NANOPARTICLES WITH BACTERIA

The scope of this section is to summarize concisely the mechanism of interaction between AgNPs and bacteria. Although this topic has been deeply investigated in many works, here we aim at giving an account on the main processes that ultimately lead to the modification of the particle optical features.

Although the complex chain of events leading to bacterial eradication is still being investigated in detail, the main antibacterial agent of AgNPs has been identified in the Ag⁺ species, while the nanoparticles themselves serve the function of silver atom reservoirs.¹⁰ Silver ions are released as a result of an oxidative dissolution reaction, which occurs thanks to the synergic effect of oxygen and a nucleophilic ligand, such as thiols, amines, and phosphates that are present in the bacterial cell envelope.¹¹ It is interesting noting that such a reaction is exploited commercially for the extraction of gold from metal ore by using cyanide as nucleophilic ligand (gold cyanidation).

The role of oxygen and oxidative processes was specifically investigated by Xiu et al.,¹² who compared the antibacterial cytotoxicity of AgNPs in anaerobic and aerobic environments. The study showed negligible effects of the AgNPs on the bacterial growth rate in the anaerobic case, despite the high particle concentration, largely above the estimated lethal concentration of Ag⁺ ions. This observation leads to conclude that the presence of oxygen is necessary for the release of Ag⁺ ions, confirming that the bacterial toxicity of AgNPs stems from the oxidative dissolution process. A detailed description of the Ag⁺ release mechanisms was already proposed in 2010,¹³ wherein one can in fact see that oxygen intermediates play a substantial role in extracting the positive ions from the particle surface.

The role of the electrostatic interaction between the positively charged silver ions and bacteria cell walls has been elucidated in the past couple of decades. In particular, several studies showed a greater growth inhibition in gram-negative than gram-positive bacteria.^14,15 This was attributed to the structure of their respective cell walls: whereas gram-negative bacteria have a layer of negatively charged and flexible lipopolysaccharides at the exterior, the cell wall in gram-positive bacteria is principally composed of a thick layer of zwitterionic and rigid peptidoglycan. The superior effectivity of Ag⁺ in inhibiting growth in gram-negative bacteria has been thus ascribed to the higher mechanical flexibility and density of negative charges in this kind of bacteria than the gram-positive counterparts.¹⁴ In these regards, the role of Ag/cell wall electrostatic interaction has been evidenced by the fact that positively charged AgNPs showed a much lower minimum inhibitory concentration value against E. coli (two orders of magnitude) with respect to their neutral counterpart.

Finally, the uptake of Ag⁺ ions occurs mostly by endocytosis, which has been argued to cause accumulation of silver in lysosomes as well as increases in the permeability of the cell membrane by peroxidation of membrane lipids, which also lead to cytoplasm leakage.^16,17 A study by Ansari et al.¹⁸ showed a gradually increasing degree of aggregation of AgNPs in the E. coli cytoplasm and loss of cytoplasmic material through the membrane as a function of AgNP concentration. In addition, the dissolution process of Ag⁺ ions not only degrades the cell membrane, but also leads to the generation of reactive oxygen species (ROS), which are a known cause of apoptosis. The degradation of the membrane combined with the biocidal action of ROS is therefore the most commonly accepted description of the bactericidal action of silver surfaces and AgNPs in particular,.^17,19,20

In Sec. III, we will discuss the most relevant bacteria-induced modification on AgNPs in terms of aggregation, shape, and charge carrier density, and how all these parameters can influence the LSPR.

III. OPTICAL RESPONSE OF SILVER NANOPARTICLES UPON INTERACTION WITH BACTERIA

In AgNPs, the collective oscillations of the metal electron density (plasma oscillations) are confined to nanoscale volume and quantized, giving rise to discrete and localized plasmon states, the so-called localized surface plasmon resonance (LSPR). The fact that such a distinctive absorption feature is extremely sensitive to change in the dielectric environment and charge carrier density makes them viable options for use in optical modulation and detection. For instance, our group has reported recently on the combined use of plasmonic nanostructures and photonic crystals for building up photo/electrochromic devices.^21–24 Specifically to AgNPs, the LSPR usually manifests as a broad absorption feature lying from the UV-visible^25,26 to near-infrared range,²⁷ depending on particle size distribution and shape.²⁸ Specifically, Valenti and Giacomelli have recently demonstrated that this parameter can be used to monitor the oxidation state of AgNPs and the degree of dissolution.²⁹ In particular, the intensity of LSPR peak decreases when the nanoparticles are exposed to an excess oxidizing agent, namely, H₂O₂. This effect occurs even in the presence of protein coatings that prevent particle agglomeration in biologically relevant conditions, thus indicating that LSPR peak damping is a reliable indicator of the oxidative dissolution process.

Along these lines, Desai et al. showed that the LSPR peak position and intensity is strongly related to the oxidation state of silver, as they could monitor the formation and the size of AgNPs via simple UV–Vis absorption measurements.³⁰ The authors observed that the plasmonic resonance of AgNPs decreases in intensity as time progresses due to spontaneous oxidation along with particle size. On the other hand, exposure to a reducing compound, in this case NaBH₄, delays the dissolution process, with relatively high concentrations of the reducing agent maintaining the AgNPs' plasmon resonance largely unchanged over a long time span.

The role of oxidative dissolution in influencing the optical properties of AgNPs has been investigated more in detail by Morgensen and Kneipp³¹ in 2014 (Fig. 1). In particular, they observed an intrinsic blue shift of the plasmon resonance in the extinction spectra of AgNPs exposed to oxidizing agents over time, which was mainly attributed to the reduction in particle size. This was preceded by a red shift when using an oxidizing substance such as cysteamine, due to the change in the refractive index. However, it is noted that the same was not observed with other media such as cyanide, the refractive index change of which is much smaller, nor with direct oxidants such as hydrogen peroxide. This study allowed disentangling between the optical effects due to change in the dielectric environment (cysteamine-induced red shift) from those related to the modification of the charge carrier density (oxidative dissolution–induced blue shift).

FIG. 1.

Time series of extinction spectra collected during dissolution of an AgNP film with 10-nm radius using (a) 1 mM cysteamine and (b) 0.1 mM cyanide. Spectra for t < 0 are measured in de-ionized water (DIW). Dashed arrows show the progression over typically 30–60 min. Reproduced with permission from K. B. Mogensen and K. Kneipp, J. Phys. Chem. C 118, 28075 (2014). Copyright 2014 American Chemical Society.³¹

Despite the fact that silver oxidative dissolution is one of the first and most important steps within the Ag/bacteria interaction process, the complex environment offered by the biological setting also plays an important role in the modification of the particle features. For instance, the presence of biological macromolecules, such as DNA, has been to influence the LSPR. Interestingly, Pramanik et al. showed that the intensity and peak position of LSPR of AgNPs (400 nm) can be taken as an indicator of the different interaction between the nanoparticles and the DNA of calf thymus (CT), Escherichia Coli (EC) and Micrococcus lysodeikticus (ML) (Fig. 2).³² The authors observed a quenching of the Ag LSRP at 400 nm in all cases. However, they could discriminate differences among the samples: while the CT DNA led to a LSPR red shift that was attributed to particles aggregation, the other two samples did not display such a behavior. Furthermore, AgNPs caused a damping of the DNA peak at 260 nm, which was substantial for the ML sample. Consequently, the ratio between the DNA and LSPR absorption amplitude varied from sample to sample, an effect that was attributed to the different binding constant between silver NPs and the DNA base pairs. This implies that AgNPs could be used not only to detect the presence of pathogens, but also to potentially discern between different bacterial strains.

FIG. 2.

(a)−(c) Absorption spectra of AgNPs in the presence of different concentrations of CT, EC, and ML DNA, respectively. (d)−(f) Linear plot obtained after plotting the difference in absorbance with DNA concentrations for calf thymus (CT, 0.02–5.5 μM), Escherichia coli (EC, 0.02–4 μM), and Micrococcus lysodeikticus (ML, 0.02–3 μM). Reproduced with permission from S. Pramanik, S. Chatterjee, A. Saha et al., J. Phys. Chem. B 120, 5313 (2016). Copyright 2016 American Chemical Society.³²

However, unlike in the controlled case of direct exposure to DNA, the process of detection must first and foremost take into account the interaction with the external barrier provided by the bacterial cell envelope. In such a framework, the effect of E. coli exposure on the LSPR peak of AgNPs was reported by Sepunaru et al. in 2015.³³ Here, the authors exploited the strong AgNPs/bacteria interaction and the bacterial-induced silver oxidative process to demonstrate a proof-of-concept for the electrochemical detection of single E. coli cells. To evaluate the nanoparticles' adhesion to bacterial cells, they monitored the LSPR peak as a function of bacterial concentration (Fig. 3), observing that the physiological KCl buffer alone caused a strong attenuation of the absorption peak due to ionic strength–induced aggregation. On the other hand, AgNPs' association to bacterial cells led to their stabilization, as indicated by the partial recovery of the LSPR peak. The small red shift noted in the Ag/E. coli samples was ascribed to several processes, such as surface plasmon coupling, aggregation between closely spaced NPs, or a capping agent replacement.

FIG. 3.

(a) Absorption spectra of 10 nM AgNPs in water (black), in 0.1 M KCl solution (brown) and in 0.1 M KCl solution containing 0.3 pM (red) or 0.15 pM (pink) E. coli cells. (Inset) Titration curve of the absorption peak (at 399 nm) of 10 nM AgNPs in 0.1 M KCl solution containing different concentrations of E. coli. (b) Absorption spectra of 2 nM AgNPs in 0.1 M KCl solution containing 0.3 pM E. coli, before and after filtration with 0.2 μm filter. This experiment permitted to confirm the relatively high association between E. coli cells and silver nanoparticles. Reproduced with permission from L. Sepunaru, K. Tschulik, C. Batchelor-McAuley et al., Biomater. Sci. 3, 816 (2015). Copyright 2015 Royal Society of Chemistry.³³

Conversely, LSPR damping in the presence of E. coli and Pseudomonas aeruginosa has been reported in a more recent paper.³⁴ This was attribute to the development of resistance to AgNPs, which stems from the production of the adhesive flagellum protein flagellin in swimming bacteria, which in turns leads to particles' aggregation and reduction of their antibacterial activity. In general, these studies provide important insights that may be exploited for bacterial optical detection.

More recently, we have shown that the spectral position of the LSPR in silver nanoplates films undergoes a blue shift upon exposure to bacteria (Fig. 4).^35,36 In particular, exposure to the culture medium only [Luria-Bertani, (LB)] leads to a clear red shift of the plasmon absorption, a result that can be attributed to the increase in the effective refractive index. On the other hand, a blue shift in the absorption band is observed after contamination with E. coli. This was ascribed to an increase in the charge carried density (bio doping), possibly owing to the oxidative dissolution process. Interestingly, contamination with the gram-positive Micrococcus luteus leads to a more convoluted effect, encompassing both a red shift due to the culture medium and an increase in the high-energy absorption (blue shift). Such an intricate readout was related to a less sensitive interaction between the silver surface and gram-positive bacteria due to the different cell wall structures. In a more detailed spectroscopic work, we have seen that such a blue shift in the linear absorption can be related to a series of events, namely, particle rounding out, shrinking, and amorphization, which stem from the oxidative dissolution process.³⁷ Integration of such a response with photonic crystals can be exploited to build up colorimetric devices for the detection of bacteria, in which the plasmon response is translated into a bacterial-induced color shift.^35,36,38 In addition, the enhanced interaction of silver with gram-negative bacteria suggests that AgNPs can be used not only as a standalone bactericidal and detection medium, but also as a way to functionalize other plasmonic surfaces. For instance, Yao et al. have reported on the functionalization of a gold surface with immobilized AgNPs,³⁹ leading to an enhancement of the plasmon resonance blue shift as a function of the E. coli concentration. This can open the way for further options and approaches available for bacterial optical sensing.

FIG. 4.

Optical absorption of 8 nm-thick silver layers composed of nanoplates on glass substrate before (silver pristine) and after exposure (a) to LB culture broth and (b) to E. coli, and before and after exposure (c) to the tryptic soy culture broth and (d) to M. luteus. The blue dashed lines represent the differential absorption expressed as (Abs_{E. coli} − Abs_pristine)/Abs_pristine. Reproduced with permission from G. M. Paternò, L. Moscardi, S. Donini et al., Faraday Discuss. 223, 125 (2020). Copyright 2020 Royal Society of Chemistry.³⁶

Conversely, it has been observed that the LSPR position and width of AgNPs can be taken as a descriptor for their antimicrobial efficacy. In particular, Mlalila et al. showed that smaller NPs exhibiting narrower LSRP perform better in terms of antimicrobial action than larger NPs with broader response.⁴⁰ In addition, they noted that positively charged AgNPs showed an enhanced LSPR, a narrower width, and superior antimicrobial activity as compared to neutral ones.⁴¹ The relatively high area-to-volume ratio of small NPs, as well as the more effective electrostatic interaction between small positive ions with the bacterial cell walls, have been taken into account to explain those findings. This study can potentially provide useful information for the optimization of the material's microbe detection sensitivity.

IV. CONCLUSIONS

The research on the interaction of AgNPs with bacteria has produced very important results in recent years, showing promising aspects that can be employed to overcome the issue of multi-drug resistance in pathogens. While a complete and accurate description of the chemical processes behind the AgNP cytotoxicity has been largely discussed, the effects of the Ag/bacteria interaction on the silver LSPR has attracted little attention to date. This is a pity, since the high sensitivity of the LSPR position, intensity, and width to the dielectric and chemical environment render them powerful optical detection tools. For instance, the oxidative dissolution process that is at the basis of the release of the Ag⁺ toxicant upon interaction with bacteria can lead to LSPR blue shift because of the increased charge carrier density. Furthermore, bacterial-induced particle aggregation causes clear damping of the LSPR, thus suggesting that this parameter can also be taken as a physical descriptor of the interaction. With the tools at our disposal, the fabrication of appropriately designed AgNPs for bacterial detection purposes is therefore possible and may lead to the development of efficient monitoring and screening medical devices for various pathogens. However, the exact photophysics behind the oxidative dissolution of AgNPs and its spectroscopical signatures are not yet univocally clear: while the plasmon shift has been noticed in most works, both blue shift or red shift is reported, seemingly depending on the specific environment in consideration. There is therefore room for further research into the photophysics and specific mechanism of the AgNPs–bacteria interactions in order to have precise knowledge of their optical response and for potential applications of such devices as versatile and effective pathogen detectors.

AUTHORS' CONTRIBUTIONS

All authors contributed to manuscript drafting and revising, and figure creation.

CONFLICT OF INTERESTS

The authors declare financial support from the company DG for Life. The authors clarify that the scientific conclusions that are presented in this study are not influenced by the activity of the company.

DATA AVAILABILITY

Data sharing is not applicable to this article, as no new data were created or analyzed in this study.