Introduction

Escherichia coli (E. Coli) and Staphylococcus aureus (S. aureus) are among the highest bacteria that have developed resistance to antibiotics (Poolman and Anderson 2018; Guo et al. 2020; van den Honert et al. 2021; Muehler et al. 2022; Hou et al. 2022). E. coli is one of the most common nosocomial pathogens that cause urinary tract infections (UTIs) (Gao et al. 2023) and enterocolitis. S. aureus is also an etiological infection agent responsible for significant levels of morbidity and mortality (Wainwright et al. 2017).

Antimicrobial photodynamic inactivation (aPDI) is an innovative process that can be employed for disinfection (Manoharan et al. 2022). aPDI utilizes a photosensitizer (PS), light and oxygen to inactivate metabolic cells (Souza et al. 2021). This acts as a foundation for designing hybrid nanoconjugates for exploitation of desirable molecular properties. The principle focuses on combining diverse properties of nanomaterials to obtain a hybrid nanoconjugate with outstanding photophysical properties compared to a single unit. In a pioneering study, Ledwaba et al. harnessed the photophysical properties of cationic 5,10,15,20-tetra(pyridin-3-yl) porphyrin and Zn(II) derivative coupled to graphene quantum dots for the photoinactivation of E. coli. (Ledwaba et al. 2022). The study yielded a total log reduction of 9.42.

The great efficacy demonstrated by aPDI in microbial inactivation is attributed to the generation of reactive oxygen species (ROS) (Meerovich et al. 2021). This phenomenon inhibits microbial resistance by targeting nucleic acids, lipids and proteins (Pereira et al. 2014; Alves et al. 2016). Research has suggested porphyrins as effective PSs in aPDI studies because of high electron and energy transfer, resistance to photobleaching, advantageous photophysical and structural properties (Malá et al. 2021; Rapacka-Zdończyk et al. 2021). However, only cationic porphyrins are effective against both Gram-positive and Gram-negative bacteria, while non-ionic and anionic porphyrins are rendered less effective against Gram-negative bacteria (Merchat et al. 1996; Banfi et al. 2006). This limitation can be addressed through conjugation of porphyrins to a semiconductor material that induces antimicrobial activity by producing free radicals that damage the cell wall (Pati et al. 2016).

Among various semiconductor materials, quantum dots (QDs) have gained more interests in antimicrobial research (Lu et al. 2008; Priester et al. 2009; Joshi et al. 2009; Ma et al. 2018). These are crystalline semiconductors of nanometer dimensions with desirable characteristics relating to conductivity, optical and electronic properties (Banin et al. 1999; Rajendiran et al. 2019). Neelgund et al. employed CdS/Ag2S QDs (2–19 nm) for inactivation of Pseudomonas aeruginosa and the investigation yielded positive results (Neelgund et al. 2012). Similarly, 3-mercaptopropionic acid (MPA)-capped CdTe QDs (1–10 nm) showed antimicrobial activity against Salmonella typhimurium (Geraldo et al. 2012). The antimicrobial mechanisms were attributed to Cd2+ disrupting cellular pathways, penetrating the cell wall and damaging the DNA structure. Due to toxicity of binary QDs comprising of Cd and Pb, research now focuses more on ternary QDs made up of group I-III-VI elements (e.g. CuInS2 (CIS)) for controlled toxicity (You et al. 2019; Zikalala et al. 2020).

In the current study, the conjugation of novel metal-free and indium(III) derivatives of 4-(15-(4-boronophenyl)-10,20-diphenylporphyrin-5-yl)benzoic acid to CIS/ZnS core–shell QDs immobilized on mesoporous silica for photoinactivation of E. coli and S. aureus is reported. The resulting nanoconjugates are variants of porphyrin-CIS/ZnS QDs-mesoporous silica nanohybrids capable of enhanced antimicrobial effects.

Experimental

Materials and equipment utilized in this study are outlined in the supporting information.

Synthesis of metal-free and In(III) 4-(15-(4-boronophenyl)-10,20-diphenylporphyrin-5-yl)benzoic acid

The porphyrins utilized in this study were synthesized using a method by Jeong et al. with modifications outlined in Scheme 1 (Jeong et al. 2010). The metal-free derivative was synthesized by dissolving 1a (2.249 g, 0.015 mol), 1b (2.173 g, 0.015 mol), 1c (3.121 g, 0.031 mol) and 1d (2.898 g, 0.043 mol) in propionic acid (100 mL) and stirred for 4 h at 200 °C. The resulting mixture was then cooled to room temperature overnight whilst stirring. Completion of the reaction was checked with UV/Vis spectrometry followed by column chromatography to obtain pure product. A mixture of silica gel and ethyl acetate was utilized as the stationary phase, while DCM and ethyl acetate [1:1] were used as the mobile phase. The eluted fractions from the column were compared for best fit UV/Vis data for deciding which fraction to crystallize from. 1M HCl was used to precipitate the metal-free porphyrin, resulting in a product that was filtered, washed with distilled water and then air-dried.

Scheme 1
scheme 1

Synthesis of metal-free and In(III) 4-(15-(4-boronophenyl)-10,20-diphenylporphyrin-5-yl)benzoic acid

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Metal insertion was then conducted to improve fluorescence of the porphyrin by favouring intersystem crossing. Metal ions with a heavy atom effect such as indium(III) are recommended (Nyokong and Antunes 2010; Ndlovu et al. 2022). This metal can limit fluorescence by promoting spin-forbidden processes and thus generating significant free radicals (Zoltan et al. 2015). Metalation took place by dissolving InCl3 (0.442 g, 0.002 mol) and sodium acetate (0.117 g, 0.002 mol) in acetic acid (50 mL). The metal-free porphyrin (0.001 mol) was then added to the mixture and refluxed for 4 h. HCl (1 M) was used for precipitation, and the product was filtered, washed with distilled water and air-dried.

POR(H2) Yield (45,53%), FTIR: –NH (3421 cm−1), –OH (3368 cm−1), –CH3/–CH2 (2894–2986 cm−1), C=O/C=C (1686–1650 cm−1), C=N (1553 cm−1), C–N (1485 cm−1), C–O (1176 cm−1), B–C (1012 cm−1). MALDI-TOF–MS m/z calc: 702.58 found [M–H] 701.8. UV/Vis (DMSO) λmax nm (log ε): 415 (5.25), 511(4.88), 550 (4.26), 598 (4.10), 650 (4.01). 1H NMR (400 MHz, DMSO-d6) δ, ppm: − 2.94 (s, 2H, NH pyrrole), 4.13 (s, 2H, B-OH), 6.07–6.57 (m, 8H, β pyrrole), 6.83 (m, 2H, Hpara phenyl), 7.06 (d, 4H, Hmeta phenyl), 7.11 (d, 4H, Hortho phenyl), 7.36 (d, 2H, Hmeta phenyl-B), 7.49 (d, 2H, Hortho phenyl-B), 7.65 (d, 2H, Hmeta phenyl-COOH), 7.86 (d, 2H, Hortho phenyl-COOH), 12.71 (s, H, COOH).

POR(In) Yield (89,61%), FTIR: = –OH (3374 cm−1), –CH3/–CH2 (2781–2948 cm−1), C=O/C=C (1660–1646 cm−1), C=N (1561 cm−1), C–N (1488 cm−1), C–O (1201 cm−1) and B–C (1008 cm−1). MALDI-TOF-MS m/z calc: 850.8 found [M + 3H]+ 853.4. UV/Vis (DMSO) λmax nm (log ε): 420 (4.96), 517 (4.11), 599 (4.05), 651 (3.94). 1H NMR (400 MHz, DMSO-d6) δ, ppm: 4.21 (s, 2H, B-OH), 6.38–6.64 (m, 8H, β pyrrole), 6.69 (m, 2H, Hpara phenyl), 7.00 (d, 4H, Hmeta phenyl), 7.18 (d, 4H, Hortho phenyl), 7.59 (d, 2H, Hmeta phenyl-B), 7.75 (d, 2H, Hortho phenyl-B), 7.91 (d, 2H, Hmeta phenyl-COOH), 7.99 (d, 2H, Hortho phenyl-COOH), 11.94 (s, H, COOH).

Synthesis of CuInS2/ZnS core/shell quantum dots functionalized with glutathione

A solvothermal method was employed for the synthesis of CIS/ZnS QDs with modifications from a reported study (Karimi et al. 2019). A Cu/In ratio of 1:2/3 was utilized. Cu(OAc)2 (91 mg, 0.75 mmol), In(OAc)3 (146 mg, 0.5 mmol) and DDT (20 mL) were added into a 100 mL Teflon-lined autoclave at room temperature. DDT was employed as a source of sulphur. The reaction mixture was then heated for 6 h at 180 °C. After nucleation, the Teflon-lined autoclave was cooled to room temperature to obtain a red product of CuInS2 QDs. This product was then coated with ZnS to improve its stability, fluorescence properties and limit toxicity. The zinc stock solution was prepared by dissolving Zn(OAc)2 (367 mg, 2 mmol) in DDT (4 mL), OA (2 mL) and ODE (8 mL) for 30 min at 160 °C. A clear solution was obtained and added to the CIS core solution. The final mixture was inserted into the Teflon-lined autoclave and further heated for 16 h at 200 °C for the shell to grow. The mixture was purified by adding chloroform and excessive acetone into the mixture multiple times to obtain pure CIS/ZnS QDs.

Functionalization with GSH was conducted by dissolving CIS/ZnS QDs (500 mg) in chloroform (5 mL) and adding GSH (200 mg, 0.65 mmol). The reaction mixture was then stirred for 24 h at 30 °C. The final product was precipitated with acetone, washed with distilled water and dried in the fume hood.

Synthesis of APTES-functionalized mesoporous silica

APTES-functionalized mesoporous silica was synthesized with modifications from a previous study (Goscianska et al. 2017). Pluronic F127 (0.80 g) and sodium dioctyl sulfosuccinate (200 mg, 0.450 mmol) were dissolved in a mixture of millipore water (30 mL) and H2SO4 (2.0 M) at 50 °C. Then, mesitylene (400 mg, 3.328 mmol) was added and the mixture was stirred until a transparent solution was obtained. After clear solution, tetraethyl orthosilicate (3.80 g, 0.018 mol) and 3-aminopropyltriethoxysilane (300 mg, 1.673 mmol) were added. The reaction proceeded for a day at 50 °C. After a day, the mixture was sealed in a 100 mL Teflon-lined autoclave and subjected to hydrothermal activity at 120 °C for another day. The resulting product was filtered under vacuum, washed with distilled water and dried. A solid product was obtained and calcined at 600 °C for 6 h to get rid of the template.

Conjugation of Porphyrin-CIS/ZnS-GSH to APTES-mesoporous silica

This procedure was conducted in two steps. Firstly, POR(H2) or POR(In) was conjugated to APTES-mesoporous silica by forming a peptide bond with –NH2 and –COOH groups. This proceeded by dissolving POR(H2)/POR(In) (0.025 g, 0.036 mol/0.025 g, 0.029 mol) into DMF (10 mL) and adding DCC (0.030 g, 0.000145 mol) to convert the –COOH group of the porphyrin into an active carbodiimide ester group. The mixture was stirred for 24 h at room temperature, and APTES-mesoporous silica (0.3 g) was added; this was allowed to stir further for another day. The conjugates were then separated from the non-conjugate particles using Bio-Beads S-X1 from Bio-Rad. The resulting nanoconjugate was Porphyrin-mesoporous silica.

The second step involved the conjugation of CIS/ZnS-GSH to the nanoconjugates of porphyrin-mesoporous silica. This was done by dissolving CIS/ZnS-GSH (0.5 g) in chloroform (5 mL) and adding DCC (0.030 g, 0.000145 mol) to convert the -COOH group of GSH into an active carbodiimide ester group. The mixture was stirred for 24 h, and POR(H2)- or POR(In)-APTES-mesoporous silica nanoconjugate was added and then further stirred for another day. This conjugation step is based on the formation of ester bonds between the –COOH groups of GSH and –OH groups (B-OH) of the porphyrin. The final products are separated with Bio-Beads S-X1, precipitated with acetone, centrifuged and air-dried.

The resulting nanoconjugates are POR(H2)-CIS/ZnS QDs-Silica (Metal-free derivative) and POR(In)-CIS/ZnS QDs-Silica (Metalated derivative). Figure 1 shows a representation of POR(H2)-CIS/ZnS QDs-Silica showing the three units (i.e. mesoporous silica, POR(H2) and CIS/ZnS QDs). GSH and APTES are also outlined as the conjugation/linking agents.

Fig. 1
figure 1

Proposed structure of nanoconjugate consisting of metal-free 4-(15-(4-boronophenyl)-10,20-diphenylporphyrin-5-yl)benzoic acid (POR(H2)), CIS/ZnS QDs and APTES-mesoporous silica

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Antimicrobial methods

Bacteria strain and culture conditions

A reported technique was utilized for the photoinactivation studies (Dalgaard et al. 1994). The microbes (E.coli or S. aureus) were incubated in 6 mL of prepared broth for 48 h at 37 ℃ in a rotary shaker with 200 rpm. A spectrophotometer at 600 nm was then used to confirm an optical density of 0.6–0.8 as for the experiments to proceed, followed by centrifuging and washing with phosphate-buffered saline (PBS) to get rid of the broth. This was conducted in triplicates at 4500 RPM for 5 min. The supernatant was decanted, and pellet was stored in 100 mL PBS (10−2 dilution factor) under refrigeration. A dilution factor of 10−5 (1 × 109 CFU/mL) was later found as the optimal bacteria concentration optimized for experiments.

Nanoconjugate

POR(H2)-CIS/ZnS QDs-Silica and POR(In)-CIS/ZnS QDs-Silica were suspended in 2% DMSO in PBS. A concentration of 10 µg/mL was used for photoinactivation studies. No effect was noted on the bacteria when a control experiment was conducted. The control is when the nanoconjugates were not included in the experiment.

Light source and exposure

The employed photoinactivation tool was an LED-mounted irradiation chamber fitted with M415L4 LED. A radiance of 15.6 μW/mm2 was delivered onto the samples. The nanoconjugate-bacteria solutions were placed in a well microplate opposite the LED, followed by irradiation with minimal to zero external light interreferences. The solutions were localized by incubating for 30 min before irradiation (Makola et al. 2020).

Results and discussion

Characterization

Nuclear magnetic resonance (NMR), mass spectrometry (MS) and Fourier-transform infrared (FTIR) spectroscopy

The porphyrin complexes were characterized using MS, 1H-NMR and FTIR. The MS analysis of POR(H2) molecular ion peaks at m/z = 701.8, which correspond to [M–H] (Figure S1 in supporting information). POR(In) revealed molecular ion peaks at m/z = 853.4, which correspond to [M + 3H]+ (Figure S2). The 1H-NMR was obtained for POR(H2) and POR(In) (Figures S3 and S4). A pyrrole NH is observed for POR(H2) at − 2.94 ppm, while it is not observed for POR(In), suggesting the successful metalation of the POR(H2). The obtained 1H-NMR data correspond to the proposed structures of POR(H2) and POR(In). FTIR was also utilized for identifying the present functional groups. Both complexes had –C–H aromatic peaks between 2850 and 2921 cm−1, followed by the C–N peak at 1581 cm−1 and B–C peak at 1012 cm−1. Notably, the stretch peaks from 3250–3500 cm−1 are observed (see Figure S5), which appear broadly on POR(H2) than it does on POR(In) suggesting the presence of both OH and NH groups in POR(H2) and only OH groups in POR(In). The overall characterization data were satisfactory for the synthesized compounds.

UV/Vis spectroscopy

Porphyrins are characteristic of notable UV/Vis absorptions in the visible and ultraviolet regions. This is depicted as weak Q bands and an intense Soret band as shown in Fig. 2. One would also notice an intense colour of porphyrins in solution or solid phase due to highly conjugated π-electron systems. The Soret bands of POR(H2) and POR(In) were observed at 415 nm and 420 nm, respectively, when dissolved in DMSO. Red shifting occurred as indium was introduced, which causes the delocalization of electrons in the porphyrin macrocycle (Giovannetti 2012). POR(H2) and POR(In) are asymmetric porphyrins with boron on one side of the molecule, and intramolecular energy transfer occurs away from boron to the rest of the molecule. This makes the porphyrin to also exhibit fluorinated boron-dipyrromethene (BODIPY)-like absorption characteristics. Typical BODIPY absorptions occur between 500 and 518 nm, while those of porphyrins occur around 601 and 649 nm (Jeong et al. 2010). POR(H2) exhibits absorption bands at 511, 550, 598 and 650 nm, while POR(In) shows absorption bands at 517, 599 and 651 nm. Notably, the observed UV/Vis spectra are characteristic of summed BODIPY and porphyrin spectra.

Fig. 2
figure 2

Absorption spectra of POR(H2) and POR(In) porphyrin derivatives in DMSO

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The photostability of PSs is a crucial aspect that is closely studied to monitor the photodegradation profile under light irradiation. This is an undesirable phenomenon that negatively impacts the optimal performance of the PS. High photostability is often possessed by PSs with low energy of HOMO. This limits the occurrence of photooxidation and photo-transformation processes (Sułek et al. 2020). Since chloro-substituted PSs are characterized by low energy of HOMOs, they are expected to be more photostable (Costa et al. 2012). The photostability of POR(H2) and POR(In) is depicted in Fig. 3, and POR(In) is the more photostable derivative. The presence of InCl3 favours the electron-withdrawing effect by promoting reduction and limiting oxidation (Hayashi et al. 2014).

Fig. 3
figure 3

The photostability of POR(H2) and POR(In) in 2% DMSO

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Redshifts are also observed in the UV/Vis spectroscopy data of the nanoconjugates (Table 1). Figure 4 shows the absorption spectra of POR(H2)-CIS/ZnS QDs-Silica and POR(In)-CIS/ZnS QDs-Silica, which indicate the absorption bands that are characteristic of the components forming the conjugates. The absorption peaks observed at 339 nm, 665 nm and close to near infrared (NIR) are descriptive of the QD absorption bands. The absorption bands of a porphyrin are noted at 400–650 nm, consisting of Soret bands and Q bands. Self-aggregation of the porphyrins was also monitored using the UV/Vis spectroscopy. A blueshift of the Soret band absorption with an increase in concentration will result in an H-aggregation. The opposite outcome, which is redshifting, will result in J-aggregation (Ding et al. 2021). Shown in Fig. 5 are the UV/Vis spectra of POR(H2) and POR(In) in increasing concentration for deducing the aggregation behaviour. The absorption peaks of both porphyrins showed no significant change in terms of the wavelengths. This means POR(H2) and POR(In) do not aggregate in DMSO at these monitored concentrations.

Table 1 The photophysical and photochemical parameters for POR(H2), POR(In), POR(H2)-CIS/ZnS QDs-Silica and POR(In)-CIS/ZnS QDs-Silica in DMSO
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Fig. 4
figure 4

Absorption spectra of POR(H2)-CIS/ZnS QDs-Silica and POR(In)-CIS/ZnS QDs-Silica compared to CIS/ZnS QDs in solid state

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Fig. 5
figure 5

Aggregation behaviour of a POR(H2) and b POR(In) in DMSO at different concentrations

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Fluorescence emission spectra

The porphyrins were dissolved in DMSO, and their fluorescence emission spectra were recorded. Excitation at 490 nm resulted in peak observations at 655 and 720 nm for POR(H2), attributed to Q00 and Q01 transitions, respectively (Soy et al. 2019). Emission peaks for POR(In) were observed at 609 and 660 nm (Fig. 6).

Fig. 6
figure 6

Fluorescence emission spectra of POR(H2) and POR(In) as examples, excited at λ = 490 nm in DMSO

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Dynamic light scattering (DLS)

The average size distribution of the nanoconjugates was deduced using DLS measurements, in Fig. 7. The CIS/ZnS QDs-Silica conjugates have a smaller size as compared to the conjugates with porphyrins. Also, the POR(H2)-CIS/ZnS QDs-Silica conjugates have a smaller size as compared to the corresponding metalated conjugate. This is influenced by alteration of hydrophobicity induced by central metals in the porphyrin cavity. Thus, larger sizes for POR(In)-CIS/ZnS QDs-Silica conjugates are observed. The sizes of 195 nm, 223 nm and 256 nm were established for CIS/ZnS QDs-Silica, POR(H2)-CIS/ZnS QDs-Silica and POR(In)-CIS/ZnS QDs-Silica, respectively. The increasing sizes are testament to the formation of peptide and ester bonds between porphyrins, quantum dots and silica.

Fig. 7
figure 7

Dynamic light scattering (DLS) distribution curve for CIS/ZnS QDs-Silica, POR(H2)-CIS/ZnS QDs-Silica and POR(In)-CIS/ZnS QDs-Silica in 2% DMSO

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X-ray diffraction (XRD)

The X-ray powder diffractogram pattern for POR(H2)-CIS/ZnS QDs-Silica and POR(In)-CIS/ZnS QDs-Silica as presented in Fig. 8 indicates polycrystallinity and monophasic character. However, the XRD pattern for mesoporous silica (in Figure S6) suggests an amorphous nature similar to POR(In) and POR(H2) in Fig. 8. Thus, the crystallinity can be attributed to the conjugation of QDs on the mesoporous silica surface.

Fig. 8
figure 8

XRD pattern of POR(H2)-CIS/ZnS QDs-Silica and POR(In)-CIS/ZnS QDs-Silica

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Scanning electron microscopy (SEM)

SEM images were used to study the morphology and particle distribution of nanoconjugates. Figure 9 shows a bulky mesoporous silica with amorphous nature covered by spherical QDs supported by porphyrins on the mesoporous silica surface, while Figure S7 only shows the mesoporous silica with notable porosity veins. In Fig. 9, CIS/ZnS QDs are observed to be attached on the mesoporous silica surface acting as a nanocarrier. Overall, both nanoconjugates showed identical morphological characteristics when SEM was utilized.

Fig. 9
figure 9

SEM images of POR(H2)-CIS/ZnS QDs-Silica (a) and POR(In)-CIS/ZnS QDs-Silica (b)

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Energy-dispersive microscopy (EDS)

Energy-dispersive microscopy (EDS) was employed for qualitative analysis of the elemental compositions of the synthesized nanoconjugates. The elemental composition of POR(H2)-CIS/ZnS QDs-Silica and POR(In)-CIS/ZnS QDs-Silica is depicted in Fig. 10a and b, respectively. The synthesized nanoconjugates show the expected elements. Peaks of Cu, In, S and Zn relate to the composition of CIS/ZnS QDs. The elemental composition of some material was vaguely detected because the surface of the mesoporous silica was covered by the QDs (Fig. 9). Hence, the detected elements are those of CIS/ZnS QDs.

Fig. 10
figure 10

EDS images of POR(H2)-CIS/ZnS QDs-Silica (a) and POR(In)-CIS/ZnS QDs-Silica (b)

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Fluorescence lifetimes (τF) and quantum yield (Ф F)

Fluorescence lifetime (τF) is the average time a fluorophore spends in the excited state prior to emitting a photon and returning to the ground state (Matarazzo and Hudson 2015). Time-Correlated Single Proton Counting (TCSPC) was used for recording the fluorescence decay curves and τF analysis. The fluorescence decay curve is shown in Fig. 11 for POR(In)-CIS/ZnS QDs-Silica as an example. The τF were determined to be 7.22, 5.67, 8.67 and 6.13 ns for POR(H2), POR(In), POR(H2)-CIS/ZnS QDs-Silica and POR(In)-CIS/ZnS QDs-Silica, respectively (Table 2).

Fig. 11
figure 11

Fluorescence decay (blue), X2 fitting (black) and IRF (red) curves for POR(In)-CIS/ZnS QDs-Silica in DMSO

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Table 2 Log reduction values for photoinactivation effect of POR(H2), POR(In), POR(H2)-CIS/ZnS QDs-Silica, POR(In)-CIS/ZnS QDs-Silica on S. aureus and E. coli in 15 min irradiation with Thorlabs M415L3 LED
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Fluorescence quantum yield (ФF) values show the ratio of photons absorbed to photons emitted through fluorescence. They depict how efficient an excited molecule returns to the electronic ground state by photon emission. This process is influenced by various parameters such as electronic structure, steric and conformational interactions (Lakowicz 2006). ФF values were obtained using a reported method from the literature where ФF = 0.039 for ZnTPP (TPP = tetraphenyl porphyrin) was used as a standard (Tran Thi et al. 1989; Ogunsipe et al. 2004). Calculations show the ФF values of the unmetalated derivatives to be higher than the corresponding metalated derivatives. Metalation was the major principle in this observation as it promotes quenching of fluorescence (Hayashi et al. 2014). The ФF values were determined to be 0.043, 0.025, 0.049 and 0.030 for POR(H2), POR(In), POR(H2)-CIS/ZnS QDs-Silica and POR(In)-CIS/ZnS QDs-Silica, respectively (Table 1).

Singlet oxygen quantum yield (Φ Δ)

Singlet oxygen generation is a principal contributing factor in the efficacy of porphyrins in photoinactivation of microbes via oxidative stress (DeRosa 2002). ΦΔ was determined in dry DMF using DMA as a singlet oxygen quencher and ZnTPP (singlet oxygen quantum yield of standard (ΦΔstd) = 0.53) as a standard (Kee et al. 2008). It is well documented that metalated porphyrins are more efficient ROS generators as compared to the metal-free analogues (Dąbrowski et al. 2015; Skwor et al. 2016). Higher ΦΔ values were observed for POR(In) compared to POR(H2) in this study as well. The ΦΔ values of POR(H2) and POR(In) were determined to be 0.43 and 0.56, respectively. After conjugation, the ΦΔ values of POR(H2)-CIS/ZnS QDs-Silica and POR(In)-CIS/ZnS QDs-Silica were determined to be 0.59 and 0.72, respectively. Notably, conjugation of porphyrin to QDs improved the singlet oxygen generation.

The Soret and Q bands of (H2) were not altered during the degradation of DMA as shown in Fig. 12, which validates the photostability of the porphyrin. A similar outcome is also observed for POR(In) as shown in Figure S8 of the supporting information.

Fig. 12
figure 12

Variations in DMA absorption spectra with irradiation time in the presence of POR(H2)

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Triplet lifetime (τ T)

For accurate determination of triplet state parameters for the conjugates, the sample is de-oxygenated to prevent quenching of the triplet state macrocycle by oxygen (Openda et al. 2022). Thus, the samples were de-gassed using argon for 1 h before recording the triplet lifetime decay curves, which is shown in Fig. 13 as an example. The obtained values were 87.9, 68.2, 40.6 and 29.7 µs for POR(H2), POR(In), POR(H2)-CIS/ZnS QDs-Silica and POR(In)-CIS/ZnS QDs-Silica, respectively. The longest τT values that were determined involved the metal-free derivatives, while the indium derivatives had shorter times due to the heavy atom effect.

Fig. 13
figure 13

Triplet lifetime plot of POR(In)-CIS/ZnS-Silica as an example

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Antimicrobial studies

The Gram (−) bacterial strain employed in this study was E. coli, while S. aureus was utilized for Gram (+) bacterial strain investigations. All photoinactivation studies were conducted using 2% DMSO in a phosphate-buffered solution (PBS). Reliability and validity of the results was strengthened by tripling the photoinactivation procedure of each sample. The antimicrobial activity of the nanoconjugates was then investigated and evaluated. A concentration of 10 µg/mL was used for photoinactivation studies. The bacteria/photosensitizer mixtures were incubated in an oven equipped with a shaker for 30 min in the dark at 37 °C before plating. 100 μL of solution was taken from the suspension and was immediately inoculated on agar plates to determine the activity at 0 min without treatment. 2.5 mL of each incubated bacterial-Por suspension was kept in the dark (dark treatment), and 2.5 mL was irradiated at the B-band maximum of the photosensitizers in a 24 well plate for 0, 5, 10,15 min of which after 15 min there were no bacteria that survived. After irradiation and dark treatment, 100 μL aliquots of each suspension was inoculated on agar plates. The number of colonies was then counted on each plate after 18 h of incubation at 37 °C. Control treatments were performed in the absence of photosensitizer both with irradiation and in the dark to determine the effect of the light and solvents on the bacteria. All experiments were carried out three times. The data for CFU/mL were converted to the logarithmic form, and reduction percentages were calculated.

A minimum log reduction of 3 Log CFU is recommended by the Food and Drug Administration (FDA) for a PS to be recognized applicable for photoinactivation (Sobotta et al. 2019). Findings from this study suggest that POR(H2) and POR(In) are not recommended for photoinactivation of E. coli strains. Log reductions of 1.47 and 1.49 log CFU were achieved for POR(H2) and POR(In), respectively, against E. coli, whereas a 9.76 log CFU reduction was achieved separately when S. aureus was subjected to both porphyrins, thus fulfilling the FDA’s recommendations in this case. This supports that non-ionic porphyrins are less effective against gram (−) bacteria and more efficient against gram (+) bacteria as detailed in our recent work (Ndlovu et al. 2022). This observation is attributed to the presence of lipopolysaccharides and sialic acid in the cell wall of gram (−) bacteria, which makes the surface anionic and inhibits electrostatic interaction to non-ionic porphyrins (Hamblin 2016). Further observations show porphyrin phototoxicity to be directly proportional to the irradiation time (Figs. 14 and 15).

Fig. 14
figure 14

Logarithmic reduction of S. aureus under irradiation (15.6 μW/mm2) with a concentration of 10 μg/mL of POR(H2), POR(In), POR(H2)-CIS/ZnS QDs-Silica and POR(In)-CIS/ZnS QDs-Silica using a Thorlabs 415 nm LED

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Fig. 15
figure 15

Logarithmic reduction of E. coli under irradiation (15.6 μW/mm2) with a concentration of 10 μg/mL of POR(H2), POR(In), POR(H2)-CIS/ZnS QDs-Silica and POR(In)-CIS/ZnS QDs-Silica using a Thorlabs 415 nm LED

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Nanoconjugates comprising of porphyrins and QDs potentially exhibit enhanced photoinactivation characteristics as compared to their individual analogues. Conjugation results in a syngenetic effect induced by the conjugate’s components (Openda et al. 2021). In a pioneering study, Magaela et al. reported improved photoinactivation when cationic 5,10,15,20-Tetra(pyridin-3-yl) porphyrin and Zn(II) derivative were conjugated to graphene quantum dots, resulting in low ФF and high Ф∆ values which are favourable parameters. Similarly, this work yielded corresponding results when POR(H2) and POR(In) were conjugated to CIS/ZnS QDs and mesoporous silica for support. POR(H2)-CIS/ZnS QDs-Silica and POR(In)-CIS/ZnS QDs-Silica achieved complete photoinactivation of S. aureus with a 8.08 and 9.76 log CFU reduction, respectively. The nanoconjugates also produced equivalent log reductions of 9.38 log CFU towards E. coli. We find that CIS/ZnS QDs could compensate the drawbacks of non-ionic porphyrins against Gram (−) bacteria. Overall, the % bacterial viability in the presence of the porphyrins and nanoconjugates is detailed in Table 3. E. coli was viable after 15 min of irradiation in the presence of POR(H2) and POR(In), but zero viability was detected when POR(H2)-CIS/ZnS QDs-Silica and POR(In)-CIS/ZnS QDs-Silica were utilized. S. aureus showed poor viability against the porphyrins and nanoconjugates. Zero viability was detected after 15 min of irradiation in the presence of POR(H2) and POR(In), and zero viability was detected after 10 min of irradiation when POR(H2)-CIS/ZnS QDs-Silica and POR(In)-CIS/ZnS QDs-Silica were utilized.

Table 3 % Bacterial viability of POR(H2), POR(In), POR(H2)-CIS/ZnS QDs-Silica, POR(In)-CIS/ZnS QDs-Silica for inactivation effect on S. aureus and E. coli in 15 min irradiation with Thorlabs M415L3 LED
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Conclusions

Nanoconjugates comprising of metal-free or In(III) 4-(15-(4-boronophenyl)-10,20-diphenylporphyrin-5-yl)benzoic acid, CIS/ZnS QDs and mesoporous silica were sequentially synthesized. Various spectroscopic techniques were used for characterization of the nanoconjugates. ФF and ФΔ quantum yields improved for porphyrins in the presence of CIS/ZnS QDs, which enhanced the aPDI performance. The study is evidence of improved photoinactivation through enhancement of ISC as a result of heavy atom effects. As such, the photoinactivation efficacy against S. aureus and E. coli was greatly improved. Moreover, conjugation of non-ionic porphyrins to nanomaterials with desirable properties is proven effective against E. coli. Lastly, utilization of mesoporous silica as a support has no negative effect on the photoinactivation efficacy of the nanoconjugate. Thus, it is graded as a satisfactory support material since complete photoinactivation is achieved.