1. Introduction
Antimicrobial photodynamic inactivation (aPDI) technology is a novel non-thermal antibacterial technology that uses reactive oxygen species (ROS) produced by light-activated photosensitizers to cause microbial death [1,2]. Studies have shown that aPDI can effectively kill bacteria such as Staphylococcus aureus (S. aureus), Escherichia coli (E. coli), and Salmonella, and that it has a significant antibacterial effect on fungi such as Aspergillus niger, Aspergillus flavus, Penicillium griseus, and Candida albicans [3,4]. In addition, its greatest advantage is that a tolerance is not easily developed in harmful microorganisms [5,6,7].
The antibacterial mechanism of aPDI is such that when light of a specific wavelength is irradiated on the microorganism, some of the light is absorbed by the photosensitizer. This becomes excited to a higher energy state, forming an excited singlet state (1PS*). As this state is unstable, the photosensitizer returns to a ground state or forms a stable excited triplet state (3PS*). The excited triplet state reacts with intracellular substrates, particularly oxygen, to transfer energy to the oxygen molecules, subsequently generating ROS [8]. ROS generation processes can be classified as Type I or Type II. ROS are key to the aPDI process, and are known to exert antibacterial effects. They generate oxidative stress and can simultaneously act on multiple targets—including destroying the cell walls and membranes of microorganisms, adversely affecting the genetic material (DNA) of cells, and interfering with the normal expressions of proteins—to synergically cause the death of pathogenic bacteria [5,7]. Therefore, the selection of the light source and type as well as control of the photosensitizer dose and ROS content should be considered to optimize the antibacterial efficiency of aPDI. Dong et al. demonstrated the influence of curcumin-mediated photodynamic technology (Cur-PDT) on Bacillus subtilis (B. subtilis), verifying that after irradiation with 120 W of blue light for 15 min, 50 μmol/L curcumin reduced B. subtilis (107–108 colony-forming units (CFUs)/mL) to undetectable levels [9]. aPDI based on sodium magnesium chlorophyll (SMC, 10 µmol/L) could reduce the number of viable S. aureus units by more than 6 log CFU/mL [10]. Yan et al. also found that the count of S. aureus gradually decreased to 4.99 log CFU/mL as the SMC concentration was increased to 5 μmol/L [11]. Yang et al. used zinc oxide nanoparticles (ZnO-NPs) as an antibacterial agent and photosensitizer, and measured their ability to inhibit Acinetobacter baumannii (A. baumannii) under blue light irradiation. The results indicated that with a ZnO-NP dose of 0.125 mg/mL and under irradiation with 5.4 J/cm2 blue light, the inhibition rate of A. baumannii was 50%. However, when the concentration of ZnO-NPs was increased from 0.5 to 1 mg/mL, the inhibition rate rose to 100% [12]. The aforementioned studies indicate that to optimize the aPDI effect, it is necessary to select materials and reagents carefully, considering the specific application.
Although aPDI, as a mature technology, is widely used in medical and clinical applications, its applicability to the field of food quality and safety remains an active research area [13,14,15,16,17]. Owing to its advantages of an adequate degermation performance, environmental friendliness, high safety, and low cost, aPDI has considerable potential in food quality and safety applications [18]. However, aPDI is not limited to a potential utilization as a food antibacterial as it may also be used for the decontamination and antibacterial treatment of distribution and kitchen equipment or packaging materials. In addition, aPDI can be used in aquaculture. The addition of photosensitizers into culture ponds for aquatic produce with irradiation can result in antibacterial effects, and may replace the use of antibiotics in aquaculture. Hence, aPDI has a significant long-term application potential in this field, and it can contribute to aquaculture ecosystem health through prevention and control as well as ensuring food quality and safety at the source [17]. In summary, harmful microorganisms are one of the main threats to food quality and safety; aPDI is an excellent countermeasure, especially for food that requires a low-temperature antibacterial treatment. In food-grade antibacterial treatments, it is necessary to use non-toxic materials. Therefore, combining materials with non-toxic properties to achieve aPDI is a topic for future research.
The toxicity of gold nanoparticles (Au NPs) is discussed in many articles in the literature; a few are toxic whereas others demonstrate no toxicity in vivo owing to their excellent biocompatibility, especially at low additive concentrations [19]. Au NPs synthesized using citrate and polyvinylpyrrolidone or other commonly used non-toxic surface coatings or capping agents generally have no antibacterial effect [20]. Shankar prepared 140 ± 13 nm Au NPs and used them in an antibacterial test with S. aureus and E. coli. They found that even with a concentration of 128 μg/mL, the Au NPs showed no antibacterial effect [20]. Although Au NPs have a poor antibacterial effect, they can enhance the antibacterial effect of other molecules and can be used as drug delivery carriers owing to their unique physiochemical properties [21].
Erythrosine is a suitable eco-friendly photosensitizer that is also used as a red pigment in food. Moreover, it has an excellent antibacterial effect and is harmless to humans [22]. Therefore, researchers have started using it as an antibacterial drug for oral biofilm treatments, and many studies have proved its excellent antibacterial effect on foodborne pathogens [23,24]. Lee et al. used an erythrosine-based photosensitive pigment in combination with a dental halogen curing unit on Streptococcus mutans, a caries-causing bacteria, for an antibacterial treatment. It was experimentally found that the bacterial death rate reached as much as 75% after 8 h, verifying the excellent efficiency of erythrosine as a photosensitizer for antibacterial treatments [25].
The wavelengths of green light correspond with the absorption band of erythrosine; hence, this type of light can maximize the antibacterial effect of aPDI. Moreover, green light can be obtained simply and at a low cost, and is harmless to the human body. The use of green light can make an experimental setup more practical. Therefore, in this study, the photosensitizer erythrosine was used with a green light source and Au NPs for antibacterial treatments.
However, to improve the aPDI performance of erythrosine in practical applications, it is essential to further enhance the aPDI effect. Therefore, we hypothesized that three non-toxic materials could effectively improve the aPDI performance; in the current study, we set out to test this hypothesis. We posited that the combination of environmentally friendly Au NPs with the edible pigment erythrosine should produce an increase in the number of photocatalysts per unit area on a bacterial surface. Energy wastage could be minimized by pairing them with a monochromatic green light source to avoid irradiation with a wavelength corresponding with the absorption band of erythrosine. It was expected that this strategy would result in a strong synergic effect to increase the aPDI performance.
In addition to an aPDI effect enhancement, the antibacterial mechanism was also explored. Under irradiation, photosensitizers exhibit Type I and Type II responses [26], which can occur simultaneously and compete with each other, depending on the specific photosensitizer properties, substrate characteristics, and oxygen concentration [26,27]. Akhtar et al. examined ROS through 2′,7′-dichlorofluorescein diacetate (DCFH-DA) staining and confirmed the type of ROS generation by fluorescence spectroscopy. Their data indicated that a large amount singlet oxygen was generated, suggesting that Type II ROS generation was the cause of the antibacterial effect [28]. The experimental results also proved the feasibility of their method in verifying the antibacterial mechanism of aPDI [20]. By understanding the antibacterial mechanisms, it is possible to select suitable materials and environments to enhance the antibacterial effect.
The aim of this study was to combine non-toxic reagents and a safe light source to optimize aPDI performance in a suitable environment. A non-toxic food coloring (erythrosine) was used as a photosensitizer. Green light, which is at the peak of the solar electromagnetic spectrum and can be considered to be harmless, was used for irradiation. In addition, Au NPs, which are non-toxic to the human body, were used as carriers [29]. The photosensitizer (erythrosine) was hypothesized to efficiently adhere to the Au NPs. Furthermore, the Au NPs were shown to have a good bacterial affinity in the inactivation process, and acted as carriers to provide adsorption sites for the photosensitizer (erythrosine) molecules. Therefore, the Au NPs and erythrosine were suggested to have a synergic effect on the inactivation performance. White light-emitting diodes (LEDs) were also used as light sources in the aPDI experiments. The emission spectrum of a white LED is composed of blue, green, and yellow light. Herein, we hypothesized that white light irradiation would similarly generate an aPDI effect; therefore, a continuous antibacterial treatment under general LED illumination for everyday applications is a realistic possibility.
Moreover, we used three scavengers to study the erythrosine inactivation mechanism, which is important to explore. We expect this knowledge will be helpful in determining the most suitable environment for future practical inactivation needs.
2. Materials and Methods
In this experiment, erythrosine (Red No. 3) was selected as the photosensitizer, and a green LED light source (525 nm) was used for the antibacterial experiment with E. coli as the target bacteria. The antibacterial effect of erythrosine with green light was explored, and Au NPs were added to the experimental system to compare the antibacterial effect with and without Au NPs. This approach was chosen for the antibacterial experiment because aPDI is a non-invasive treatment that specifically targets without causing additional damage to the surrounding tissues.
The E. coli used in the experiment were cultured in a lysogeny broth and placed in a shaking incubator at 37 °C for 15 h. Erythrosine was selected as the photosensitizer and a green LED module with a wavelength of 525 nm was selected as the light source because erythrosine absorbs light with a wavelength of 526 nm. The module consisted of 24 LEDs, and a cooling fan was added to the LED light panel to prevent bacterial death due to the system overheating. The Au NPs were chemically prepared with sodium citrate (Na3C6H5O7·2H2O, Panreac Applichem, Germany) as the reducing agent and tetrachloroauric(III) acid (HAuCl4·3H2O, Acros Organics, NJ, USA) as the raw material [30].
The photosensitizer (erythrosine) had a concentration of 0.631 mM, and the green LEDs had a power setting of 7.2 W. Au NPs of four different sizes (10, 20, 30, and 40 nm) synthesized via chemical reduction were used. First, the colonies were only irradiated with green light. Erythrosine (20 μL) and a bacteria suspension (50 μL) were mixed in an Eppendorf tube without green light irradiation to observe the bacterial survival; the mixture was then exposed to green light for different periods of time (1, 2, 3, 6, and 9 min) to observe the associated bacterial survival. Subsequently, 20 μL of a Au NP solution (containing 1.6 × 10−3 g Au) was added to the system, which was then exposed to green light for different time periods (1, 2, 3, 6, and 9 min). After each aPDI treatment, the number of CFUs was counted, and a LIVE/DEAD fluorescent reagent was used to repeatedly test and confirm the bacterial survival via confocal laser scanning microscopy (LSM 780, Carl Zeiss, Oberkochen, Germany). Scanning electron microscopy (SEM, Auriga, Carl Zeiss, Oberkochen, Germany; voltage: 15 kV) configured to possess an energy dispersive spectroscopy (EDS) capability was used to observe the adhesion of Au NPs on the surface of the E. coli cells. Finally, a ROS experiment was performed by adding different types of scavengers to study the mechanism of E. coli death. Ascorbic acid, mannitol, and sodium azide (Sin Fang Liang Chemical Original Instrument Co., Ltd.) were used as scavengers with an identical concentration of 10 mM. The following procedure was used. A total of 1 μL of 10 mM DCFH-DA (ThermoFisher Scientific, Waltham, MA, USA) was added to 1000 μL of PBS. The aPDI-treated bacteria were washed twice with PBS, following which the precipitated bacteria were evenly mixed with DCFH-DA and then mixed in the incubator at 37 °C for 30 min. The bacterial suspension was centrifuged at 10,000× g for 10 min. Subsequently, the bacterial body was washed twice with PBS and allowed to stand in a 37 °C incubator for approximately 20 min. The bacteria were then dropped onto a glass slide, which was covered with an 18 mm coverslip, and allowed to stand for approximately 15 min. The prepared samples were observed with an inverted fluorescence microscope (Carl Zeiss, Oberkochen, Germany). Details of the conditions and labeling codes for the control and experimental groups are summarized in Table 1.
3. Results and Discussion
Figure 1 shows the appearances of Au nanoparticles with four different sizes. The average sizes of the Au nanoparticles were (a) 11 nm, (b) 20 nm, (c) 29 nm, and (d) 40 nm. For the convenience of explanation in the subsequent discussions, all the wavelengths were marked at 10 nm, 20 nm, 30 nm, and 40 nm.
The CFU plate counting method was used to determine bacterial survival after aPDI. The results obtained were accurate, verifying the reliability and universal applicability of this method. Figure 2 shows the CFU results of the aPDI-treated systems that were irradiated with green light for 60, 120, 180, 360, and 540 s with and without the addition of Au NPs of different particle sizes. Clearly, the addition of Au NPs enhanced the aPDI performance. However, using CFUs for visual observations has limitations. Therefore, we investigated the aPDI performance by analyzing the bacterial death rate.
Figure 3 shows the CFU plate counts and mortality rates. After 1 min of aPDI treatment, the mortality rate of E. coli in the (PS+, Au−, L+) group was approximately 33% whereas in the (PS+, Au+, L+) group, it was approximately 60%. After 3 min of aPDI treatment, the mortality rate of E. coli in the (PS+, Au−, L+) group was approximately 58% whereas in the (PS+, Au+, L+) system, it was approximately 80%. Finally, after 9 min of aPDI treatment, the death rate in the (PS+, Au−, L+) and (PS+, Au+, L+) groups reached 97%. Table 2 lists the detailed colony statistics. These demonstrate that adding Au NPs could effectively increase the death rate, especially for the shorter exposure times. Figure 2 and Figure 3 and Table 2 illustrate that the inactivation performance was better with Au NPs than without Au NPs. Meanwhile, experiments were conducted using Au NPs of different sizes; these results indicated that the larger the size of the Au NPs (40 nm), the better the inactivation performance. This may have been because the larger Au NPs could provide more adsorption sites for the photosensitizers. Therefore, the efficiency of the antibacterial treatment was improved. The Au concentration used in this study was relatively low. Thus, the aPDI effect, which originated from the ROS generated by the interaction of the photosensitizers and green light, could not be observed in the (PS+, Au−, L−), (PS−, Au+, L−), and (PS−, Au−, L+) groups.
Figure 4 shows the results of the LIVE/DEAD bacterial viability tests. The green and red fluorescence signals corresponded with the presence of live and dead bacteria, respectively; hence, this assay allowed a clear visualization of the bacterial survival. Figure 3 shows the results for the untreated group (PS−, Au−, L−) and the results for the (PS+, Au−, L+) and (PS+, Au+, L+) groups after the aPDI treatment. A comparison of the results of the latter two groups indicated that erythrosine with green light irradiation could cause the death of E. coli; adding Au NPs could effectively improve the antibacterial efficiency. The death rate was obtained by analyzing the fluorescence images, as shown in Table 3. The bacterial death rate of the untreated bacteria (PS−, Au−, L−) was approximately 2.71%. Using erythrosine with green light (PS+, Au–, L+) significantly increased the death rate to 9.14%. This phenomenon confirmed the aPDI effect of erythrosine. After adding Au NPs (PS+, Au+, L+), the bacterial death rate after aPDI increased nearly three-fold compared with that of the group without Au NPs, demonstrating that Au NPs and erythrosine had a synergic effect and effectively improved the aPDI performance.
SEM was conducted to understand the aPDI-treatment-induced morphological changes in E. coli (Figure 5). Figure 5a shows an SEM image of E. coli before the aPDI treatment. A contoured appearance with wrinkles on the surface of the E. coli was apparent. However, after the aPDI treatment, the E. coli cells appeared shrunken and broken (Figure 5b). This indicated that the aPDI treatment destroyed the integrity of the bacteria and led to bacterial death. Figure 5c shows the surface of E. coli after aPDI with residual Au NPs, indicating that the aPDI-treated E. coli could not maintain its original shape, resulting in severe damage. We hypothesized that Au NPs could be used as carriers for the photosensitizer and would have a high affinity to E. coli [31]. Therefore, Au NPs were expected to have a synergic effect on the aPDI performance. Before irradiation, SEM was used to observe the surface morphology of the bacteria (PS+, Au+ (40 nm), L+) group at a high magnification. The visible particles shown in Figure 5d were speculated to be Au NPs. Many Au NPs could be observed on the E. coli cells, indicating that the nanoparticles could be effectively adsorbed onto the bacterial surface.
To verify this hypothesis, a qualitative elemental analysis was conducted using EDS. The results in Figure 6 indicated that the bacterial surface contained Au. Therefore, it could be confirmed that the particles seen on the bacterial surface in Figure 5d were Au NPs. It is known that Au NPs have no independent antibacterial effect [14]. When a substance is reduced to nanometer-scale particles, the number of surface atoms increases relative to the total number of atoms. This results in increased surface energy and more vital van der Waals forces; hence, interactions with other molecules are formed more readily. It was, therefore, speculated that the high surface energy Au NPs in this experiment assisted with the adherence of the photosensitive molecules to the bacterial surface, increasing the overall contact surface area and the probability of contact between the photosensitizer and bacteria, thereby enhancing the antibacterial activity via a synergic effect. This speculation also explained why the antibacterial effect in the (PS+, Au+, L+) group was more significant than that in the (PS+, Au−, L+) group.
A direct observation of ROS generation is a straightforward approach to further understand whether adding Au NPs can improve the aPDI performance. In this study, the generation of ROS was detected using DCFH-DA. If ROS were present, the bacteria would appear fluorescent green; the higher the ROS concentration, the stronger the fluorescence. The results, shown in Figure 7, showed that fluorescence appeared after green light irradiation and erythrosine addition, and became significantly stronger after adding Au NPs. This indicated that the high E. coli death rate was correlated with the high ROS concentration. Simultaneously, when the size of the Au NPs increased from 10 to 40 nm, the green fluorescence became more apparent. Based on the above evidence (Figure 5d, Figure 6 and Figure 7), it could be suggested that the greater number of photosensitizer molecules on the surface of the larger Au NPs generated more ROS after interacting with the light. This finding also validated the hypothesis that Au NPs could be used as photosensitizer carriers.
The average gray value can be calculated using a black/white image. The color scale between black and white is divided into several levels represented by a numerical value between 0 and 255, with 0 representing black and 255 representing white. This scale can be used to distinguish brightness. The smaller the numerical value, the less bright the image; the lower the fluorescence intensity, the lower the ROS levels. The cumulative fluorescence intensity can, therefore, be calculated as the sum of the gray values in a fluorescence image; a greater value indicates a stronger fluorescence and higher ROS levels. Figure 8 indicates that the average gray values and fluorescence intensities in the (PS+, Au+, L+) group were higher than in the (PS+, Au–, L+) group. In addition, when the size of the Au NPs increased from 10 to 40 nm, the fluorescence intensity significantly increased, indicating a higher number of ROS.
Ascorbic acid can quench the hydroxyl radical (OH·) [32] and singlet oxygen (1O2) [33], mannitol can quench the hydroxyl radical (OH·) [8], and sodium azide can quench singlet oxygen (1O2) [8]. The fluorescence images obtained after adding these scavengers to those that had been E. coli-treated with the photosensitizer and irradiated are shown in Figure 9. The cells to which mannitol was added retained a strong green fluorescence signal, indicating that the ROS generated by the combination of the green light and erythrosine were not hydroxyl radicals. In contrast, the cells that added ascorbic acid or sodium azide did not emit green light, indicating that ROS were absent from these samples. Ascorbic acid and sodium azide had apparent inhibitory effects on the ROS, implying that the death of the E. coli bacteria was because of the existence of singlet oxygen species (Type II response). The results of this study were consistent with those of previous studies [34]. However, the scavenger used in this study did not quench the O2− radical; therefore, these results provided no information on whether or not O2− radicals were produced under these aPDI conditions.
4. Conclusions
This study investigated the synergic effect of erythrosine and Au NPs in aPDI. Using erythrosine with green LED irradiation resulted in an antibacterial effect in an aPDI experiment used on E. coli. Simultaneously, although Au NPs alone have no antibacterial effect, their use with erythrosine, as in the (PS+, Au+, L+) aPDI treatment group, could enhance the antibacterial effect for a given treatment time. The SEM observation indicated that the high surface energy of the Au NPs enabled the photosensitizer to be adsorbed on their surface. Au, therefore, served as a carrier to ensure that the photosensitizer came into contact with the bacteria, resulting in a synergic effect that enhanced the antibacterial effect. It was tentatively concluded that the more substantial aPDI enhancement by the larger Au nanoparticles was because of their greater surface area.
In addition, the experiments showed that the generation of ROS caused bacterial death. The amounts of ROS detected in the (PS+, Au+, L+) group were more significant than in the (PS+, Au−, L+) group. Moreover, adding sodium azide (singlet oxygen scavenger) and ascorbic acid significantly reduced the ROS fluorescence signal. By contrast, the addition of mannitol did not lead to significant changes, indicating that the death of E. coli was due to the generation of singlet oxygen, and that aPDI occurred via a Type II ROS generation mechanism. This study revealed the possibilities for enhancing the antibacterial effect of aPDI treatments by selecting suitable materials and environments. aPDI, novel non-thermal antibacterial technology, presents a solution to control harmful microorganisms in food, which is of great significance for maintaining food quality and safety. Although this study investigated the aPDI performance of E. coli alone, many bacterial strains can cause food contamination. Hence, the findings of this study will aid in future aPDI research on various strains of bacteria.
Conceptualization, S.-C.S. and S.-W.Y.; methodology, S.-C.S. and S.-W.Y.; validation, S.-C.S. and F.-I.L.; formal analysis, S.-C.S. and S.-W.Y.; investigation, S.-C.S. and S.-W.Y.; resources, S.-C.S. and F.-I.L.; data curation, S.-W.Y. and Y.-C.X.; writing—original draft preparation, S.-W.Y. and Y.-C.X.; writing—review and editing, S.-C.S. and F.-I.L.; supervision, S.-C.S.; project administration, S.-C.S.; funding acquisition, S.-C.S. All authors have read and agreed to the published version of the manuscript.
Not applicable.
Not applicable.
Data are available in a publicly accessible repository.
The authors gratefully acknowledge the support of the Core Facility Center of National Cheng Kung University. iEGG and Animal Biotechnology Center from The Feature Area Research Center Program within the framework of the Higher Education Sprout Project by the Ministry of Education, Taiwan, R.O.C. (MOE-111-S-0023-F).
The authors declare no conflict of interest.
Footnotes
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Enlarge this image.Figure 1. Shapes and sizes of gold nanoparticles characterized as having diameters of (a) 10 nm, (b) 20 nm, (c) 30 nm, and (d) 40 nm.
Enlarge this image.Figure 3. Bacterial mortality rate of each group with different green light irradiation times.
Enlarge this image.Figure 4. LIVE/DEAD test results for (a) (PS−, Au−, L−), (b) (PS+, Au−, L+), (c) (PS+, Au+ (10 nm), L+), (d) (PS+, Au+ (20 nm), L+), (e) (PS+, Au+ (30 nm), L+), and (f) (PS+, Au+ (40 nm), L+) treatment groups.
Enlarge this image.Figure 5. SEM images of the E. coli morphology in the (a) (PS−, Au−, L−), (b) (PS+, Au−, L+), and (c) (PS+, Au+, L+) treatment groups; (d) Au NPs adhered to the surface of E. coli.
Enlarge this image.Figure 6. EDS detection results for E. coli bacteria treated with erythrosine and Au NPs.
Conditions and labeling codes for the control and experimental groups.
| PS−, Au−, L− | No treatment, allowing natural bacterial growth |
| PS−, Au−, L+ | Influence of only green light irradiation on bacteria |
| PS−, Au+, L− | Influence of only Au NPs on bacteria |
| PS−, Au+, L+ | Influence of Au NPs and green light irradiation on bacteria |
| PS+, Au+, L− | Influence of erythrosine and Au NPs on bacteria |
| PS+, Au−, L+ | Influence of erythrosine and green light irradiation on bacteria |
| PS+, Au+, L+ | Influence of erythrosine, Au NPs, and green light irradiation on bacteria |
Detailed colony statistics for (PS+, Au−, L+) and (PS+, Au+, L+) groups.
| Time (s) | Condition | CFU | AVG | SD | Death Rate (%) | p-Value (× 10−3) | ||
|---|---|---|---|---|---|---|---|---|
| 0 | PS−, Au−, L− | 436 | 413 | 416 | 422 | 10.20 | 0 | - |
| 0 | PS−, Au+, L− | 428 | 419 | 430 | 426 | 5.86 | 0 | |
| 60 | PS+, Au−, L+ | 303 | 286 | 255 | 281 | 19.87 | 33.28 | 0.88718 |
| PS+, Au+ (10 nm), L+ | 168 | 191 | 166 | 175 | 11.34 | 58.50 | 0.02169 | |
| PS+, Au+ (20 nm), L+ | 170 | 178 | 166 | 171 | 4.988 | 59.37 | 0.00632 | |
| PS+, Au+ (30 nm), L+ | 191 | 152 | 184 | 172 | 14.42 | 59.13 | 0.03726 | |
| PS+, Au+ (40 nm), L+ | 134 | 120 | 150 | 135 | 12.25 | 68.06 | 0.01416 | |
| 120 | PS+, Au−, L+ | 247 | 243 | 236 | 242 | 4.54 | 42.61 | 0.02216 |
| PS+, Au+ (10 nm), L+ | 140 | 167 | 144 | 150 | 11.897 | 64.35 | 0.01653 | |
| PS+, Au+ (20 nm), L+ | 149 | 118 | 123 | 130 | 13.589 | 69.17 | 0.01710 | |
| PS+, Au+ (30 nm), L+ | 138 | 132 | 133 | 134 | 2.624 | 68.14 | 0.00270 | |
| PS+, Au+ (40 nm), L+ | 135 | 113 | 130 | 126 | 9.416 | 70.12 | 0.00724 | |
| 180 | PS+, Au−, L+ | 187 | 217 | 181 | 195 | 15.748 | 53.75 | 0.06891 |
| PS+, Au+ (10 nm), L+ | 108 | 86 | 87 | 94 | 10.143 | 77.79 | 0.00552 | |
| PS+, Au+ (20 nm), L+ | 60 | 72 | 66 | 66 | 4.898 | 84.35 | 0.00153 | |
| PS+, Au+ (30 nm), L+ | 87 | 62 | 95 | 81 | 14.055 | 80.71 | 0.01009 | |
| PS+, Au+ (40 nm), L+ | 66 | 81 | 77 | 75 | 6.342 | 82.29 | 0.00215 | |
| 360 | PS+, Au−, L+ | 95 | 95 | 97 | 96 | 0.942 | 77.31 | 0.00146 |
| PS+, Au+ (10 nm), L+ | 54 | 34 | 57 | 48 | 10.208 | 88.84 | 0.00333 | |
| PS+, Au+ (20 nm), L+ | 54 | 51 | 41 | 49 | 5.557 | 88.46 | 0.00141 | |
| PS+, Au+ (30 nm), L+ | 36 | 49 | 60 | 48 | 9.809 | 88.54 | 0.00308 | |
| PS+, Au+ (40 nm), L+ | 41 | 45 | 37 | 41 | 3.265 | 90.28 | 0.00094 | |
| 540 | PS+, Au−, L+ | 10 | 10 | 18 | 13 | 3.771 | 97.00 | 0.00075 |
| PS+, Au+ (10 nm), L+ | 3 | 9 | 7 | 6 | 2.494 | 98.50 | 0.00061 | |
| PS+, Au+ (20 nm), L+ | 1 | 2 | 4 | 2 | 1.247 | 99.45 | 0.00054 | |
| PS+, Au+ (30 nm), L+ | 2 | 3 | 5 | 3 | 1.247 | 99.21 | 0.00054 | |
| PS+, Au+ (40 nm), L+ | 0 | 2 | 3 | 2 | 1.247 | 99.60 | 0.00053 | |
Fluorescence image analysis results.
| Conditions | Green | SD | Red | SD | Death Rate: |
|---|---|---|---|---|---|
| PS−, Au−, L− | 361.6 | 29.85 | 9.8 | 2.86 | 2.71 |
| PS+, Au−, L− | 341.4 | 21.69 | 3.8 | 2.04 | 1.11 |
| PS−, Au−, L+ | 205.4 | 14.58 | 2.4 | 1.51 | 1.16 |
| PS−, Au+ (10 nm), L− | 254.2 | 30.56 | 6.0 | 2.23 | 2.36 |
| PS−, Au+ (20 nm), L− | 273.4 | 12.48 | 4.2 | 1.92 | 1.53 |
| PS−, Au+ (30 nm), L− | 346.0 | 10.58 | 6.8 | 3.27 | 1.96 |
| PS−, Au+ (40 nm), L− | 296.0 | 6.44 | 5.2 | 3.27 | 1.75 |
| PS−, Au+ (10 nm), L+ | 273.6 | 20.62 | 7.2 | 3.56 | 2.63 |
| PS−, Au+ (20 nm), L+ | 539.4 | 25.11 | 7.6 | 4.39 | 1.40 |
| PS−, Au+ (30 nm), L+ | 307.4 | 20.98 | 3.8 | 1.92 | 1.23 |
| PS−, Au+ (40 nm), L+ | 324.0 | 25.94 | 6.2 | 1.92 | 1.91 |
| PS+, Au−, L+ | 260.2 | 12.15 | 23.8 | 3.11 | 9.14 |
| PS+, Au+ (10 nm), L+ | 248.4 | 20.03 | 42.2 | 7.98 | 16.98 |
| PS+, Au+ (20 nm), L+ | 270.4 | 6.76 | 48.8 | 7.91 | 18.04 |
| PS+, Au+ (30 nm), L+ | 222.0 | 29.29 | 43.6 | 9.12 | 19.63 |
| PS+, Au+ (40 nm), L+ | 127.8 | 10.63 | 33.8 | 7.75 | 26.44 |
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; Shu-Wen, Yang 1 ; Yu-Chen, Xu 1 ; Fu-I, Lu 2
1 Department of Mechanical Engineering, National Cheng Kung University, Tainan 70101, Taiwan
2 Department of Biotechnology and Bioindustry Sciences, College of Bioscience and Biotechnology, National Cheng Kung University, Tainan 70101, Taiwan; The iEGG and Animal Technology Center, National Chung Hsing University, Taichung 40227, Taiwan