Synthesis of meso-tetra-(4-sulfonatophenyl) porphyrin (TPPS₄) – CuInS/ZnS quantum dots conjugate as an improved photosensitizer

Introduction

Singlet oxygen generation and detection has gained much interest within the cancer research field due to its cytotoxic nature and subsequent role in the eradication of cancer/tumor cells.¹ Amongst the many applications of this gaseous inorganic molecule such as the photo-oxidation of toxic molecules and photo-production of chemical intermediates, photodynamic therapy (PDT) still appeals as the most prominent application.^2,3 Several preparative chemical procedures have been reported for the generation of singlet oxygen; eg, thermolysis of 3,3,4,4-tetramethyl-1,2-dioxetane⁴ and photosynthesis via plant production.⁵ However, photochemical methods, whereby oxygen is irradiated by organic dyes and sensitizers viz. methylene blue and porphyrins, have been shown to produce higher quantities of the molecule.¹ Porphyrins are macro-cyclic molecules that are constituted by four pyrrole subunits that are interconnected by methylene bridge. They are highly conjugated systems with excellent photoelectric and spectroscopic properties.³

Their inherent ability to generate singlet oxygen upon irradiation with light of specific wavelength and low dark cytotoxicity has resulted in their application in cancer treatment modalities such as PDT.^6,7 In PDT, porphyrins are able to generate singlet oxygen via energy transfer in the excited triplet state to the ground state (triplet) oxygen. Their singlet oxygen efficacy, however, can be affected by environmental factors such as solvent, temperature, moiety attachments and the presence and proximity of quenching groups. Metal-free, water-soluble and highly stable porphyrins such as meso-tetra-(4-sulfonatophenyl) porphyrin (TPPS₄) have been studied for their singlet oxygen quantum yield (SOQY), tumor tissue affinity, retention times and water solubility.^8–10 Clinical studies on TPPS₄ have shown its potential as a PDT photosensitizer; however, its biological progression has been hampered by the atypical reliance of its photo-physical properties on environmental factors as listed above.

In addressing these inherent shortcomings of TPPS₄ and porphyrins at large, much research has focused on conjugated systems for their biological applications.^8,9,11 Moreover, conjugation of TPPS₄ has shown to improve the QY of QDs and disaggregation of aged QDs which allows for a wider application range of TPPS₄–nanomaterial conjugates.⁹ However, binary quantum dots (QDs) adopted to conjugate with porphyrins have added much distress due to their toxic nature attributed to the presence of heavy metals such as Cd and Pb.¹² Consequently, researchers have focused on the use of ternary QDs such as CuInS₂/ZnS and AgInS₂/ZnS QDs for biological applications. The distinctive optical properties of CuInS-based QDs have resulted in its application in a number of industries and applications such as photovoltaic cells, white light-emitting diodes, bio-medical labeling and imaging.^13,14 Due to the excellent optical properties of these ternary QDs, the conjugation of TPPS₄ to these QDs will allow the use of TPPS₄ at excitation wavelengths wherein TPPS₄ alone cannot be used. Moreover, the conjugation of QDs to TPPS₄ is expected to produce a conjugate system with the optical advantages of the QDs while maintaining the treatment efficacy of TPPS₄ thereby addressing the inherent challenges of QDs and porphyrins when applied alone in cancer treatment. In this study, glutathione (GSH)-capped CuInS/ZnS QDs were conventionally synthesized using a hydrothermal one-pot process at Cu:In (1:4). The GSH-capped QDs were conjugated to water-soluble TPPS₄ porphyrin via sulfonamide bond formation. The effect of pH, temperature and the ratio of porphyrin:QDs on the optical properties of TPPS₄ and conjugation was evaluated. Furthermore, the study evaluated the conjugates ability to generate singlet oxygen in the presence of 1,3-diphenylbenzofuran (DPBF) as a singlet oxygen scavenger under spectro-fluorometer laser light at 535 nm. DPBF was selected as a sensitizer due to its ability to rapidly react with dioxygen only and not with super-oxides or ground state (triplet) molecular oxygen.¹⁵ TPPS₄ was able to produce singlet oxygen which increased up to three times upon conjugation with highly fluorescent CuInS/ZnS QDs. The as-synthesized materials were characterized by ultraviolet detection (UV), photoluminescence (PL), dynamic light scattering (DLS), transmission electron microscope (TEM), scanning electron microscope (SEM) and nuclear magnetic resonance (¹H NMR).

Materials and methods

Chemicals

Copper chloride, indium chloride, sodium citrate, L-glutathione, sodium sulfide, zinc acetate, thiourea, benzaldehyde, propionic acid, pyrrole, sodium bicarbonate, ethanol, hydrochloric acid, dichloromethane, methanol, ethyl acetate, n-hexane, chloroform, acetone, di-sodium hydrogen phosphate buffer, petroleum ether, calcium chloride, sulfuric acid, methylene blue, 1,3-diphenylbenzofuran and dimethyl sulfoxide were purchased from Sigma Aldrich. All chemicals were of analytical grade and used without any purification except pyrrole which was distilled prior to use. Deionized water was used for all the aqueous solution preparation.

Synthesis of TPPS₄–CuInS/ZnS conjugates

The synthesis of CuInS core and CuInS/ZnS core/shell (S1), TPPH₂ (S2) and TPPS₄ (S3), is extensively reported in the supplementary information. The TPPS₄ was conjugated to GSH-capped CuInS/ZnS QDs via sulfonamide bond as shown in Scheme 1. Briefly, 2 mL of TPPS₄ (1 mg/10 mL H₂0) was added to 2 mL of CuInS/ZnS (1 mg/10 mL H₂O) and stirred for 1 hr at room temperature. The effect of temperature on the conjugation was evaluated by varying the temperature (29°C and 50°C) at which the conjugation was performed. The effect of QDs:porphyrin ratio on the optical properties was evaluated by adding different aliquots of the QDs to TPPS₄ porphyrin to obtain a different ratio of TPPS₄/QDs between 1:1 and 1:5.

Scheme 1 Schematic diagram showing the conjugation of CuInS/ZnS and meso-tetra-(4-sulfonatophenyl) porphyrin (TPPS4).

Singlet oxygen generation of meso-tetra-(4-sulfonatophenyl) porphyrin

Singlet oxygen generation was determined by the Adarsh et al¹⁹ method. Briefly, 1,3-diphenylbenzofuran (DPBF) solution (0.8 mg/25 mL DMSO), methylene blue (MB) solution (0.5 mg/25 mL DMSO), TPPS₄ solution (0.20 mg/20 mL DMSO) and CuInS/ZnS–TPPS₄ conjugate (1 mg/10 mL) were prepared. Then, 1:1 mixture of DPBF:TPPS₄, DPBF:MB, DPBF:TPPS₄-CuInS/ZnS conjugate was irradiated at 535 nm (excitation at an absorption wavelength where TPPS₄ has minimum absorption and maximum absorption for QDs) using spectro-fluorophotometry laser light for 500 s. The decrease in the intensity of DPBF was monitored at 471 nm. The SOQY was calculated against methylene blue (0.5 mg/25 mL DMSO) as a standard (SOQY =0.52) using the following formula:

1

where SOQY and SOQYref are the SOQY of the samples (TPPS₄, conjugate) and the reference (methylene blue, 0.52), m_c and m_ref are the slope of the samples and reference, A_c and A_ref are the absorbance of the samples and the reference at irradiation wavelength.

Characterization

The synthesized TPPS₄, CuInS/ZnS QDs, CuInS/ZnS–TPPS₄ conjugate were characterized using Fourier Transform infrared spectroscopy (FTIR) (Spectrum), two UATR spectrometer (Perkin Elmer, UK), photoluminescence (PL) (RF-6000, Shimadzu, Japan), ultraviolet–visible spectrophotometry (UV–vis) (Perkin Elmer UV–Vis Lambda 25 spectrometer, UK), Nuclear Magnetic Radiation (NMR, 500 MHz Bruker spectrometer); Zeta potential analyses were performed using Anton Paar Litesizer 500 (Austria). TEM was done by using JEOL JEM-3010 electron microscope operating at 200 kV.

Results and discussion

Structural and optical properties of CuInS and CuInS/ZnS quantum dots

CuInS-based QDs were synthesized hydrothermally (Figure S1) using glutathione as a surface ligand to stabilize the as-synthesized material. Glutathione is tripeptide comprised of three amino acids (cysteine, glycine and glutamic acid), and its backbone structure, therefore, consists of thiol (–SH) group²⁰ from the cysteine, which was used as a binding group to form GSH-capped CuInS/ZnS QDs. From the glutamic and glycine of GSH, the –COOH and amine groups are available for bonding with other moieties. In this study, FTIR was used to confirm thiol bonding (–SH) on the surface of the CuInS/ZnS QDs. The bands at 3127 cm⁻¹ and 1595 cm⁻¹ on the GSH spectra represent the N-H and –C=O functional groups, respectively. The shift to 1577 cm⁻¹ of the –C=O, the enhancement in peak intensity and broadening of the N–H band, and the disappearance of the –SH peak at 2519 cm⁻¹ in the CuInS/ZnS QDs confirm the formation of GSH-capped CuInS/ZnS QDs (Figure 1A).

Figure 1 (A) FTIR spectra of GSH and GSH-capped CuInS/ZnS QDs, (B) PL spectra of CuInS QDs core and CuInS/ZnS QDs core\shell, (C) PL spectra and (D) absorption spectra of CuInS\ZnS QDs core-shell at different Zn:Cu + In ratio.

While the optical properties of QDs lie in the core, the core of QDs is reported to be highly reactive and unstable. Shell passivation with a suitable material is often required in order to improve on these inherent properties while simultaneously improving their photo-stability.¹³ In this study, ZnS was used to form a type 1 band alignment with the CuInS QDs core and thus reduced any lattice mismatch between the core and the shell material. In-situ passivation of the core was performed by injecting Zn(O₂CCH₃)₂(H₂O)₂ and CH₄N₂S – as zinc and sulfur precursors, respectively. Over-coating of the CuInS QDs core with ZnS shell resulted in improved peak intensities and symmetry. The enhancement in the PL intensity is attributed to the effective passivation of the core while the slight blue shifting in the emission position (CuInS QDs em: 721 nm and CuInS/ZnS QDs em: 709 nm) is attributed to the inter-diffusion of the Zn into the core (Figure 1B). The Zn: Cu + In ratio was examined during shell formation in order to minimize the blue-shift observed in the PL spectra of the CuInS/ZnS QDs. Different concentrations of Zn precursor were added during the coating while the Cu:In feeding ratio was kept constant at 1:4. As shown in Figure 1C, a reduction in Zn concentration resulted in a hyper-chromic and red-shifting of the emission peak position (713 nm, 716 nm and 721 nm for Zn at 0.435 mmol, 0.218 mmol and 0.109 mmol, respectively). This red-shifting can be attributed to the reduction of surface defects and stabilization of the QDs structure subsequently leading to radiative recombinations. This is further supported by the improved QY at lower Zn content and thus confirm the reduction in the surface-related recombination trap states after shell passivation. Similar observation has been reported by Zhang et al.²¹ Figure 1D shows the absorption spectra of the CuInS/ZnS. A red-shift in the absorption band-edge was obtained with increasing concentration of Zn in the shell suggesting a decreasing trend in the band-gap.

Figure 2 (A, B) TEM images of CuInS/ZnS QDs, SEM of CuInS QDs (C) and CuInS/ZnS QDs (D) and the EDS of CuInS QDs (E) and CuInS/ZnS QDs (F).Abbreviations: TEM, transmission electron microscopy; QDs quantum dots ; SEM, scanning electron microscopy; EDS, electron dispersive spectroscopy.

The morphology and average particle size of the as-synthesized CuInS/ZnS corresponding to [Zn²⁺] =0.200 mmol was estimated from the TEM micrograph (Figure 2A and B). From the TEM, it can be seen that the particles are mono-dispersed and nearly spherical in shape. The average particle sizes of the as-prepared CuInS/ZnS core/shell QDs were estimated to be ~3–3.5 nm. The presence of lattice fringes (Figure 3B) confirmed the crystallinity of the material. Figure 2C and D shows the scanning electron microscope (SEM) of the core/shell, highlighting the selected area that was evaluated for electron dispersive spectroscopy (EDS). The presence of tiny holes on the surface of the CuInS/ZnS QDs was not observed in the CuInS QDS, indicating the difference in surface morphology between the CuInS core and CuInS/ZnS core/shell. The elemental composition of the as-synthesized CuInS core and CuInS/ZnS was characterized using EDS as shown in Figure 2E and F, respectively. From the EDS of the CuInS core (Figure 2E), the elemental composition consisted of Cu, In and S while the EDS of the CuInS/ZnS core/shell (Figure 2F) showed the presence of Cu, In, S and Zn further confirming the formation of the CuInS/ZnS core/shell. The presence of the C was due to the carbon grid used and the O was accounted to the GSH capping.

Structural and optical properties of meso-tetra-(4-sulfonatophenyl) porphyrin

meso-tetra-(4-sulfonatophenyl) porphyrin (TPPS₄) was synthesized using meso-tetraphenylporphyrin (TPPH₂) as the precursor as seen in S1 and S2. The formation of water-soluble TPPS₄ from TPPH₂ was confirmed using FTIR, UV and ¹H-NMR (Figure 3). FTIR spectra (Figure 3A and B) show characteristic peaks of TPPH₂ at 3315 cm⁻¹, 1474 cm⁻¹ and 1443 cm⁻¹ assigned to N–H str, –C=C str and C=Nstr of the phenyl, respectively. Upon acidification of the TPPH₂, a medium broad peak at 3436 cm⁻¹ was formed and assigned to N–H_str overlapping with O–H_str of absorbed water by the hygroscopic-sulfonated porphyrin. The sharp peak at 1419 cm⁻¹ and a medium band at 1595 cm⁻¹ are assigned to N–H bend and C–N str frequencies, respectively, which overlapped with C=Cstr of phenyl ring. Sharp bands at 1178, 1118 and 1026 cm⁻¹ are attributed to S–O str, confirming the formation of TPPS₄.^16,17,22 The changes in the optical absorption spectra from organic precursor TPPH₂ to water-soluble TPPS₄ are shown in Figure 3C and D, respectively. The changes are characterized by a shift in the UV bands of TPPH₂ (Soret: 417 and Q-bands: 514, 549, 589, 647) in comparison to TPPS₄ (Soret: 412 and Q-bands: 515, 551, 579, 633). ¹H-NMR characterization (Figure 3E) of TPPH₂ gives chemical shifts at 8.09 and 8.22 (doublet) corresponding to β-pyrrole protons, doublet at 7.58 and 7.72 ppm assigned to the ortho and meta phenyl protons. A decrease in intensity and shielding in the β-protons of TPPS₄ are observed at 8.09 ppm and 8.22 compared to TPPH₂. The presence of N–H proton signal peaks at −2.66 and −2.39 ppm for the TPPH₂ and TPPS₄, respectively, and the observed shift in the signals confirm that free base porphyrins (TPPS₄) were synthesized from organic TPPH₂.

Figure 3 FTIR spectra of (A) TPPH2, (B) TPPS4; absorption spectra of (C) TPPH2 in DMSO, (D) water-soluble TPPS4; 1H NMR spectra of (E) TPPH2 and (F) TPPS4.Abbreviations: FTIR, Fourier-transform infrared spectroscopy; NMR, nuclear magnetic resonance.

Effect of pH on TPPS₄ optical properties

Spectral changes in the absorption and emission spectra of TPPS4 (in water) at different pH values (pH 8.5 and 3.5; adjusted using phosphate buffer and hydrochloric acid) are shown in Figure 4. In a relatively alkaline medium (pH 8.5), TPPS₄ exhibited a typical etio-type porphyrin spectrum (Figure 4A) with Soret band at 412 nm and four Q-bands at 515, 552, 579 and 633 nm. The presence of all four Q-bands suggested that a mono-protonated form of TPPS₄ was synthesized in agreement with the ¹H NMR. In acidic medium, a red-shift in the Soret bands to 433 nm and the presence of only three Q-bands at 546, 593 and 645 nm was observed. This indicates the formation of one-dimensional ordered arrangements called J-aggregates²³ characteristic of di-protonated (H⁺)₂ TPPS₄ in line with reported studies.²⁴ It has been reported that the presence of nitrogen atoms in TPPS₄ permits the protonation of the two pyrrolic nitrogens of TPPS₄ thereby changing the spectra and energetic properties of the porphyrin. This was observed in the fluorescence studies (Figure 4B) of TPPS₄ as a single peak in acidic medium which altered into double peaks in alkaline medium. The two emission peaks at 643 and 703 nm (at pH 8.5) are characteristic of free-base porphyrins in the near-neutral pH region.²⁵

Figure 4 Effect of pH on (A) UV absorption and (B) Photoluminescence  (PL) emissions of TPPS4.

In acidic medium, TPPS₄ is reported to show decreased quantum yield (QY) triplet state formation and altered electronic absorption spectra with increased Stokes-shifts in the fluorescence spectra²⁵ as observed in this study (Table 1). Consequently, pH 8.5 was selected for further analysis.

Table 1 Effect of pH on the stokes shift of TPPS4

Effect of excitation wavelength on TPPS₄ optical properties

The fluorescence spectra of TPPS₄ at different excitation wavelengths is depicted in Figure 5A. Identical emission bands were observed at all the excitation wavelengths with two emission peaks at 643 and 703 nm. The obtained fluorescence results suggested that the emission position of the porphyrin is independent of the excitation energy. The ability of TPPS₄ to emit at wavelengths lower than excitation (at scan rate 6000 nm/min) wavelength suggest that it can undergo non-linear optical processes characteristic of materials with two photon emission property. Maximum emissions were obtained upon excitation at 413 nm, which was consequently selected as the excitation wavelength of TPPS₄.

Figure 5 (A) Photoluminescence (PL) spectra of TPPS4 at different excitation wavelengths, (B) PL spectra and (C) absorption spectra at different CuInS/ZnS:TPPS4 ratios and (D) radar chart of CuInS/ZnS–TPPS4 conjugate at different synthetic temperatures (insert: area chart of the conjugate).

Conjugation of TPPS₄ porphyrin with CuInS/ZnS QDs

To evaluate the binding affinity of the porphyrin on the QDs surface, systematic titration of QDs to porphyrin was performed. The evolution of the emission and absorption spectra of TPPS₄ as a function of added CuInS/ZnS QDs is shown in Figure 5B and C. This was performed by keeping the amount of TPPS₄ constant in the reaction and increasing the amount of CuInS/ZnS QDs. The PL spectra (Figure 5B) at different QDs concentrations show that with increasing QDs concentration, the optical properties of the conjugate tend toward the QDs; a similar trend is observed in the absorption spectra (Figure 5C). Due to the conjugate instability at increased QDs concentration with changes in optical spectra away from the porphyrin, ratio, 1:1 was selected as optimum ratio for CuInS/ZnS:TPPS₄ to maintain the spectroscopic properties of both the porphyrin and QDs within the conjugate.

Effect of reaction temperature and precursor ratio on the conjugation of CuInS/ZnS–TPPS₄

The conjugation reaction was performed at two temperatures (29°C and 50°C) as sulfonamide bond formation is highly dependent on temperature. As previously mentioned, the CuInS/ZnS QDs–TPPS₄ conjugate maintained the spectral properties of both the porphyrin and QDs; this was observed at both temperatures at which the conjugation was performed. Consequently, a radar chart (Figure 5D) was used to determine the fingerprint of each variable (CuInS/ZnS, TPPS₄, CuInS/ZnS-TPPS₄) in order to trace the distinct differences in the optical patterns of the as-synthesized material. From the radar chart, we were able to separate the individual optical patterns of each material. The spectra were over-layed in order to specifically distinguish where and how different the conjugates (CuInS/ZnS-TPPS₄ at 29°C and 50°C) are from the precursors (CuInS/ZnS and TPPS₄). At 29°C, the conjugate fully encompassed the spectral properties of the QDs (as it completely covered the shape of the QDs) while maintaining the spectral shape of porphyrin. However, at 50°C, the conjugate inclined toward the TPPS₄ porphyrin (yellow and blue fingerprint) while shifting away from the QDs spectra. This suggests that at reduced temperature, the conjugate formed encompasses both the QD and porphyrin at a greater capacity than at 50°C. Furthermore, from the insert (Figure 5D), the stacked curves show the relative contribution of the QDs and porphyrin to the conjugate. This further confirms that at 29°C, more of the conjugate is likely to be formed as it covers more area than at 50°C. Thus, the synthesis of the conjugate was performed at 29°C.

The electro-kinetic potential of TPPS₄ and CuInS/ZnS QDs were measured and found to be −36.4 mV and −74.8 mV, respectively. The negative charge on TPPS₄ is attributed to the sulfonate phenyl groups in its structure^11,26,27 while the net negative charge on the surface of the QDs suggests some degree of deprotonation on its surface. This implies a low probable electrostatic attraction between QDs-GSH groups and TPPS₄ molecules. However, TPPS₄ has four sulfonyl groups available for binding and CuInS/ZnS has amine groups which when combined (sulfonyl and amine group) would aid the formation of sulfonamide bond between TPPS₄ and CuInS/ZnS via the negatively charged oxygen of the sulfonate and positive charge of the amine on the GSH-capped QDs (S1). Furthermore, the high affinity of the porphyrin π-conjugated systems for metal surfaces⁹ could further foster the formation of the sulfonamide bond. To further confirm the conjugate formation, FTIR analysis was done on the purified and dried CuInS/ZnS QDs, TPPS₄ and the dried TPPS₄–CuInS/ZnS conjugate. A shift from the CuInS/ZnS QDs and TPPS₄ characteristic bands is seen in the conjugate as the O–H band shifted to 3376 cm⁻¹ from 3435 cm⁻¹ of TPPS₄. The bands at 1584 cm⁻¹ in the conjugate is assigned to –C=O bands observed at 1570 cm⁻¹ in the GSH-CuInS/ZnS QDs. Furthermore, the presence of the S=O str at 1401 cm⁻¹ band in the conjugate (absent in the CuInS/ZnS QDs spectrum) further confirm the formation of the conjugate (Figure 6).

Figure 6 Fourier-transform infrared spectroscopy (FTIR) of TPPS4, CuInS/ZnS and TPPS4–CuInS/ZnS conjugate.

Singlet oxygen generation

In this study, singlet oxygen (¹O₂) detection was determined via photochemical method using DPBF as a ¹O₂ trapping agent and methylene blue as a reference. DPBF was chosen as singlet oxygen acceptor because of its ability to rapidly react with ¹O₂ chemically. Figure 7 represents the kinetics of the singlet oxygen generation as a function of illumination time versus intensity relative to the decay rate of DPBF (at 421 nm) upon scavenging the singlet oxygen generated. The reaction rate constants (k (s-1)) were found to be −0.825±0.068 and −0.69±0.08 for TPPS₄ and CuInS/ZnS-TPPS₄ conjugate, respectively, as estimated from the slope of regression curve of Figure 7A-B. This indicates that the reaction of the conjugate in the presence of DPBF is much faster than that of TPPS₄, implying that the singlet oxygen is generated more rapidly by the CuInS/ZnS–TPPS₄ conjugate compared to TPPS₄ porphyrin alone. Compared to the synthesized material, the blank (DPBF) reaction rate was constant (Figure 7C) over the measurement intensity range suggesting a higher stability in solution. The kinetic process of the ¹O₂ generation in the presence of DPBF was presumed to be via the reaction shown below:^1,28–30

From Equation 1, the singlet oxygen generation of the material was calculated. TPPS₄ singlet oxygen was measured to be 0.19 which increased to 0.69 upon conjugation with CuInS/ZnS QDs, which is in agreement with the reaction rate constant of the conjugate. The significant increase in the singlet oxygen generation is attributed to the spectral overlap (Scheme S1) of the TPPS₄ absorption with fluorescent emission of the CuInS/ZnS as a donor thereby suggesting possibility of Forster Resonance Energy Transfer (FRET) between CuInS/ZnS QDs and TPPS₄.

Figure 7 Singlet oxygen detection of (A) TPPS4, (B) CuInS/ZnS–TPPS4 conjugate and (C) 1,3-diphenylbenzofuran (DPBF) alone (inserts: changes in emission spectra of DPBF in presence of (A) TPPS4, (B) conjugate and (C) DPBF alone).

Conclusion

In this study, we successfully synthesized meso-tetra-(4-sulfonatophenyl) porphyrin (TPPS₄)–CuInS/ZnS QDs conjugate as an improved photosensitizer. The GSH-capped CuInS/ZnS QDs were synthesized at different Zn:Cu + In ratio during shell formation in order to minimize the blue-shift observed in the PL spectra of the CuInS/ZnS. The emission profile of the QDs improved with a hyper-chromic shift in the PL peaks upon Zn passivation. The photosensitizer- TPPS₄ was synthesized using organically soluble TPPH₂ as a precursor. The as-synthesized TPPS₄ gave characteristic UV absorption spectra in alkaline medium (pH 8.5) whereas at acidic medium (pH 3.5), three Q-bands were obtained consequently, pH 8.5 was used. CuInS/ZnS–TPPS₄ conjugate was formed via sulfonamide bond and the ratio of QDs:porphyrin in the conjugate was optimized by varying the amount of QDs in the conjugate. Based on UV, PL and radar chart, a ratio of 1:1 (QDs:porphyrin) was found to be the optimal for the conjugate formation. The effect of temperature on the synthesis of the conjugate showed that optimum temperature for the conjugate synthesis is 29°C. Furthermore, conjugation of the synthesized GSH-CuInS/ZnS to water-soluble TPPS₄ porphyrin proved to enhance the singlet oxygen generation of the porphyrin as the SOQY increased from 0.19 for TPPS₄ alone to 0.69 upon conjugation. This was supported by the increase in the reaction rate constant of the conjugate compared to TPPS₄ porphyrin alone. Thus, the as-synthesized meso-tetra-(4-sulfonatophenyl) porphyrin (TPPS₄)–CuInS/ZnS QDs conjugate can be used as an improved photosensitizer in many bio-applications.