A study of microbially fabricated bio-conjugated quantum dots for pico-molar sensing of H₂O₂ and glucose

Abstract

Quantum dots (QDs) as bio-detectors have been intensively explored owing to their size dependent optical properties and are still envisioned to be used in a plethora of biomedical and healthcare areas. However, the medical application of the biosensors demands the ultrasensitive detection of the analytes, which is usually limited for the conventional methods of colorimetric and fluorescence detection. The Fluorescence Resonance Energy Transfer (FRET) process, exploited by QDs, translates the close association between the analyte and the detector into optical properties and thus promises the effective detection of biomolecules. FRET based detection systems for biomolecules utilize surface-functionalized QDs which are usually modified post production using different organic groups. In this work, a novel protocol was formulated to produce bio-functionalized QDs with controlled chemical and optical characteristics. Here, we demonstrate the first-ever biological green synthesis of MoS₂ QDs using Pseudomonas aeruginosa. The bio-functionalized QDs show green luminescence with a quantum yield of 42%, supporting their application as an optical sensor. These QDs are utilized to detect the pico-molar concentration of glucose, which makes them ideal for early diabetes detection and many biomedical applications. Also, the ability to sense pico-molar levels of H₂O₂ opens the path for its utilization in apprehending the plant signaling pathways under stress conditions.

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1. Introduction

Glucose sensing remains an important research area as its excess, as well as deficiency, has serious consequences on human health. Its accurate detection has a large number of applications and drives multi-billion dollar biomedical industries.^1,2 The future of highly selective wearable glucose biosensors depends on making them economically viable with a high level of reproducibility and mechanical and thermal stability.³ Along with glucose, hydrogen peroxide (H₂O₂) is also a naturally occurring by-product of glucose oxidation in cells, and it can easily damage the cells which is a major risk to human health. By the Fenton reaction, due to the generation of hydroxyl radicals (HO˙) and unbalanced state of glucose, it can lead to health problems such as cytotoxicity, hypoglycemia, hyperglycemia, etc. Therefore, the quantitative determination of H₂O₂ and glucose contents in biological samples and human blood is being extensively investigated. Few detection methods that are being widely used are optical sensing, colorimetry, fluorescence, etc.^4–6

The development of sensitive and selective biosensors is the crux of basic biomedical and clinical diagnosis. QDs, photoluminescent semiconductor nanocrystals, as a result of their tunable optical and physical properties offer superior biosensing over conventional methods.^7,8 QDs exhibit a high quantum yield (QY), symmetric photoluminescence, and a large Stokes shift which makes them effective probes in medical applications.⁹ For detecting biomolecules, plenty of QD surface modification strategies are practiced which include lipid encapsulation and organic-ligand exchange.¹⁰ The surface modification allows the functionalization of the QD surface which promotes the formation of dangling bonds. The surface bonds conjugate with particular biomolecules, facilitating their detection by translating the binding to changes in the optical or physical properties of QDs. The coupling of biomolecules to the QD surface is achieved through covalent or non-covalent methods that activate or incorporate functional groups on QDs.⁷ The surface groups are activated post QD synthesis via small ligand exchange, polymer or lipid encapsulation, and biomolecule conjugation of QDs. However, the biological synthesis of QDs circumvents the post-synthesis surface modification and attains surface groups during QD formation. Microbially reinforced fabrication of QDs is hypothesized to be a facile route to achieve biofunctionalized QDs.

MoS₂ QDs are reported for their use as glucose sensors via glucosidase coupling to the QDs.¹¹ Non-enzymatic detection of glucose requires the binding of the molecules to the prepared materials, which is inevitably restricted by the chemicals and thermal-based methods of MoS₂ synthesis.^12–14 Similarly, during signal transduction in plants, H₂O₂ oxidizes the cysteine based receptors and hence shows an affinity for MoS₂ QDs.¹⁵ Microbially synthesized QDs have been hypothesized to manifest a non-mediator based detection system for H₂O₂ signaling molecules. This will make them useful for understanding the ROS signaling in stressed plants. To date, several studies have been carried out where MoS₂ QDs act as sensing elements for glucose and H₂O₂. However, these studies do not demonstrate high sensitivities.

In the current work, we demonstrate the extracellular synthesis of monodisperse bio-functionalized MoS₂ QDs, which was carried out by a facultative anaerobe Pseudomonas aeruginosa (as shown in Fig. 1(a)). Here, the optical response of bio-functionalized MoS₂ QDs, in the presence of glucose and H₂O₂, was investigated using the resonance energy transfer mechanism. The bio-based system of QD production required sodium molybdate dihydrate as the Mo source while L-cysteine hydrochloride was utilized by the bacterial cells to produce sulphide.

Fig. 1 (a) Schematic presentation of step wise MoS₂ synthesis by P. aeruginosa, (b) UV-Vis absorption spectra obtained for [Mo]: x = 0.5 mM, y = 2 mM and z = 6 mM, (c) growth phase vs. MoS₂ QD absorption spectra of the media at different phases of bacterial growth i.e. x = lag, y = initial-log, and z = late-log phase, (d) cysteine concentration vs. UV-Vis absorption spectra for different Cys·HCl concentrations i.e. x = 0.05%, y = 0.2 and z = 0.6%, (e) time based absorption spectra of the precipitates for t₁ = 0 min; t₂ = 1 h; t₃ = 3 h and t₄ = 9 h, and (f) plausible mechanism for MoS₂ QD formation by a bacterial cell (the cell has been divided into two parts with and without [Mo]). (A) Cysteine·HCl imported to the cells via membrane transporters, (B) cysteine binding with cysteine desulfhydrase enzyme to produce sulfate, (C) sulfate exported outside the cell in the media, and (D) molybdenum quenching by sulfate ions to produce MoS₂ QDs.

2. Experimental section

2.1 Bio-synthesis of MoS₂ QDs

Molybdenum tolerance of Pseudomonas aeruginosa was estimated by adding molybdenum to the cell culture in a concentration of 0.5–10 mM. Samples were withdrawn after 24 h and analyzed for cell viability and cell growth. Controls were also prepared with no molybdenum addition. The optimized Mo concentration was used for MoS₂ QD synthesis. During the experiment, cysteine supplemented actively growing P. aeruginosa cells (growth and culture conditions in the ESI†) were inoculated with 1 mM Na₂MoO₄. A reddish-brown culture was obtained within 12 hours. The culture was centrifuged at 8000 rpm for 10 min and the supernatant was collected. The impact of the growth parameters and precursor concentration on MoS₂ optical properties was also estimated. The impact of cell age on MoS₂ formation was established by recording the absorption spectrum of the growth culture at each phase of cell growth. The effect of the sulphur source i.e. cysteine concentration on MoS₂ production was measured by recording UV spectra when the cells were grown in the media with a gradient concentration of cysteine i.e. 0.01–0.5%. Time-regulated MoS₂ synthesis was evaluated by recording the absorption spectra at different time intervals post culture inoculation to Mo containing media.

2.2 Characterization of the microbially prepared MoS₂ QDs

The bio-manufactured MoS₂ QDs were characterized using powder X-Ray Diffraction (XRD) with monochromatic nickel filtered Cu Kα radiation in the 2θ range of 20 to 80°. The FTIR analysis of MoS₂ QDs was carried out using a PerkinElmer spectrometer in the range of 4000 to 400 cm⁻¹. The as-prepared QDs were imaged using a JEOL 2200FS electron microscope and the samples were prepared onto a thin film of amorphous carbon supported by a copper grid. QDs were observed using Atomic Force Microscopy (AFM, Agilent Technologies, USA, Model 5100) where cells were dried on a glass substrate and scanned to obtain high-resolution micrographs of the cells with QDs. All the measurements were carried out in air at room temperature (25° C) using a silicon nitride tip (PPP-NCL) in the intermittent contact mode. The Raman spectra of MoS₂ QDs were acquired with an excitation wavelength of 532 nm (Witec UHTS 300). The chemical composition of MoS₂ QDs was studied by using X-ray photoelectron spectroscopy (PHI 5000 Versa Probe II, ULVAC-PHI, Inc., Japan) with an incident Al K_α X-ray energy of hν = 1486.6 eV. To study the optical characterization of MoS₂ QDs, UV-absorbance spectra of the MoS₂ QDs and cell culture media were obtained on a UV-1800 spectrophotometer, SHIMADZU, Japan. The band gap was estimated using Tauc plots generated from the absorption spectra with the assumption of a direct bandgap for MoS₂. The emission spectra, collected using a Xenon lamp (450 watt) with a monochromator (Gemini 180), were used to measure QDs' PL spectra at room temperature. The PL data were recorded using a CCD detector (Hamamatsu), conjugated with a monochromator (Horiba Jobin-Y von iHR30).

3 Results and discussion

3.1 Biosynthesis of MoS₂ QDs

As this is the first-ever report of MoS₂ QD synthesis by bacteria, the study requires to comprehend every mode of interaction between molybdate salt and the bacterial cell to establish an optimized protocol. There are reports regarding the bactericidal performance of MoS₂,¹⁶ and hence the effect of the excess to optimized metal ion concentration on P. aeruginosa cells needed to be probed. Sodium molybdate dihydrate salt solution (0.5–10 mM) was added to P. aeruginosa cells to estimate the tolerance threshold. Generally, molybdenum is transported into the bacterial cells to regulate enzyme synthesis but at higher concentrations, it inhibits the sulfate reduction process which makes it toxic to cells. Cells have been observed to reduce toxic Mo⁴⁺ to insoluble MoS_2.¹⁷ In the present study, the remediation method, carried out by using Pseudomonas, has been hypothesized to involve extracellular cell wall binding, confirmed by the reduction of molybdenum ions into MoS₂ QDs in the growth media. Here, no molybdenum toxicity was observed for up to 6 mM molybdate concentration (Table S1†). The effect of molybdenum concentration on MoS₂ formation has been demonstrated by plotting absorption versus Mo concentration (Fig. 1(b)). Increasing the molybdenum salt concentration more than the threshold value (≥5 mM) leads to the absence of a peak at 460 nm in the spectra, indicating no QD formation. During MoS₂ synthesis, 1 mM Na₂MoO₄ was inoculated to the cells and the bacterial colonies were found to remove 85% of the molybdenum from the media (Table S2†) in the form of brown-colored colloids identified as MoS₂ QDs (Fig. 2(a)). The biological quenching of the salt was typically fathomed out by quantifying the effect of the cell growth parameters and substrates on MoS₂ formation. The influence of the cell growth phase on the UV-Vis spectrum of the culture is shown in Fig. 1(c). It is clear from the curve that MoS₂ QDs commenced from the late log phase (∼7–8 h) of bacterial growth, manifested by an absorption peak at 460 nm. Cysteine, the sulphur source, has been found to directly impact MoS₂ formation and this impact has been determined by preparing experimental media with varying concentrations (0.05–0.6 (w/v %)) of the Cys·HCl (shown in Fig. 1(d)).

Fig. 2 (a) Cell culture centrifugate (i) yellow coloured P. aeruginosa cell culture and (ii) brown coloured MoS₂ QDs, (b) X-ray diffractogram of microbially synthesized MoS₂ QDs, (c) FTIR spectra of MoS₂ QDs, (d) particle size distribution of MoS₂ QDs, (e) comparative bar chart demonstrating the dispersity for different MoS₂ QD preparation methods, i.e. A – chemical vapor deposition; B – hydrothermal; C – multicycle microfluidic process; D – bipolar-electrode electrochemical scissoring and E – present method (P. aeruginosa mediated), (f) TEM micrographs with SAED for lattice characterization (inset), (g) magnified HR-TEM image of the distributed MoS₂ QDs (size ∼ 6 nm), and (h) EDX spectra indicating Mo and S peaks.

There was a predictable MoS₂ QD formation on increasing cysteine concentration. However, the accelerated amount of cysteine had adverse effects on P. aeruginosa due to cysteine toxicity which has resulted in limited cell growth and consequently less MoS₂ formation.¹⁸ The absorption spectra of the cells were recorded at different time intervals 0 h, 1 h, 3 h, and 9 h after Mo addition. From the spectra in Fig. 1(e), the characteristic absorption peak was found to occur at ∼9 h beyond which there was no further change in the absorption pattern. The occurrence of the other peak at 460 nm with increasing time indicated the formation of MoS₂ QDs with a sizeable lateral dimension. The plausible mechanism of MoS₂ QD formation is shown in Fig. 1(f) as validated and suggested by the spectra and other optimization experiments.

3.2 Physical and optical characterization study of the prepared MoS₂ QDs

The crystal structure of the prepared QDs was analyzed using XRD (shown in Fig. 2(b)). The peaks at 39.5° and 62°, corresponding to the (103) and (008) planes of MoS₂, were observed (JCPDF no.: 37-1492). The intense peak at ∼20° could be attributed to the carbon in the organic molecules present in the bacterial culture. The surface functional groups of MoS₂ QDs were studied using FTIR measurements (shown in Fig. 2(c)). The peak at 3443 cm⁻¹ can be ascribed to the O–H stretching bond present as absorbed water on the MoS₂ surface. Weak peaks which were observed at 465 cm⁻¹ and 650 cm⁻¹ signified the Mo–S vibrations¹⁹ and C–S stretching bonds respectively whereas peaks at 1237 and 2353 cm⁻¹ are assigned to the organic sulphate group and CO₂ respectively.²⁰ The presence of functional groups can be attributed to the media components and cell metabolites produced and released during bacterial growth.

The surface modification due to organic groups, –OH, –NO₂, –SO₂, and sulphur bonds, renders the prepared QDs promising for extremely sensitive biosensing and enhances their affinity for biomolecules such as glucose. The particle size distribution of the MoS₂ QDs, as measured from the TEM image (Fig. 2(f)), revealed the monodispersity of the QDs (shown in Fig. 2(d)). Further, MoS₂ size distribution, obtained for the current study, was compared with the dispersity achieved by the other methods of MoS₂ QD preparation (Fig. 2(e)).^21–24 From the bar chart, it can be inferred that the present method can be successfully utilized in producing small-sized MoS₂ QDs. The size distribution of the prepared QDs is impacted by the presence of biological entities on their surface which further reinforces ligand detection. The formation of crystalline MoS₂ QDs is further confirmed using Selected Area Electron Diffraction (SAED) patterns (inset Fig. 2(f)) showing a concentric circle, which is in parity with (103) reflection of MoS₂. The HRTEM image of the MoS₂ QDs, in Fig. 2(g), showed well-resolved lattice fringes, which implied a highly crystalline nature of the QDs. The lattice spacing value obtained from HRTEM was 2.3 Å, which corresponds to the (103) lattice plane of MoS₂.²⁵ The EDX spectrum demonstrated the expected Mo and S peaks (Fig. 2(h)).

In the current study, the as-prepared MoS₂ QDs were visualized using AFM. The obtained surface roughness profile is shown in Fig. 3(a). The thickness of MoS₂ QDs was ∼2 nm, suggesting the formation of few-layered QDs, which is well consistent with the high-resolution TEM images. To further confirm the formation of layered MoS₂ QDs, the Raman spectrum of the sample was obtained and is shown in Fig. 3(b). The spectrum revealed two prominent Raman peaks at 385 cm⁻¹ and 409 cm⁻¹ referring to E¹_2g (in-plane vibrations of the Mo–S bond) and A_1g (out of plane vibrations of S atoms) modes, respectively. The E¹_2g and A_1g modes of vibrations in the as-prepared MoS₂ QDs had a blue and red shift of 1 cm⁻¹ respectively as compared to bulk MoS₂ (E¹_2g and A_1g modes of vibrations at 484 and 410 cm⁻¹).

Fig. 3 (a) AFM micrograph exhibiting the QD thickness, (b) Raman spectrum of the microbially produced MoS₂ QDs, (c) XPS spectra of MoS₂ QDs, (d) UV-Vis absorption spectrum of MoS₂ QDs and cell culture media (dashed line) with a Tauc plot (inset), and (e) PL emission spectra for the QDs. The inset shows the digital photograph of MoS₂ QDs under a 365 nm UV lamp.

It caused a reduction in the frequency difference between two peaks, indicating the mitigation in the number of layers for the microbially produced MoS₂ QDs.²⁶ The surface states and compositions of resultant MoS₂ QDs were confirmed by the X-ray photoelectron spectroscopy (XPS) measurement. In Fig. 2(c), the corresponding peaks of Mo⁴⁺ 3d_5/2 at 229.3 eV and Mo⁴⁺ 3d_3/2 at 232.6 eV confirm the formation of MoS₂ QDs. The S 2p peak shows a doublet due to S 2p_1/2 (163.5 eV) and S 2p_3/2 (162.4 eV) electrons. The S 2p spectrum is fitted by using a Gaussian–Lorentzian function after baseline correction. UV-visible absorption spectroscopy was utilized to investigate the optical absorption properties of MoS₂ QDs. The bulk MoS₂ produces the characteristic absorption peaks at 340, 430, 590, and 650 nm where the direct transition from the valence band to the conduction band is ascribed to the peaks at 340 and 430 nm.²⁷ In the case of the as-prepared MoS₂ QDs, the peaks were obtained at ∼360 and 460 nm as compared to the UV-Vis spectrum of the bacterial culture (Fig. 3(d)). The optical bandgap of the as-prepared MoS₂ QDs was found to be ∼2.0 eV, calculated by using the Tauc plot (Fig. 3(d), inset), which is significantly higher than that of bulk MoS₂ (1.2 eV) which is due to the quantum confinement effect.²⁸ The Photoluminescence (PL) emission spectrum of the synthesized MoS₂ QDs was also measured and is shown in Fig. 3(e). It showed strong emission bands at 430 and 515 nm. A digital photograph of the aqueous solution of MoS₂ QDs under 365 nm excitation further confirmed the strong green emission of the sample (inset of Fig. 3(e)). The calculated PL quantum yield (QY) of the sample was found to be 46% (details in the ESI†), which further confirmed the quantum confinement effect of QDs. The PL emission band around 530 nm is attributed to the surface passivation effect of the MoS₂ QDs and the presence of surface dangling bonds and functional groups which actively contribute to the electron transfer process. Furthermore, we have observed another PL emission band at ∼420 nm. This could be linked to the presence of the carbon structure originating during the biological synthesis process of MoS₂ QDs.

3.3 Bio-synthesized MoS₂ QDs as a H₂O₂ and glucose sensor

The green PL emission of the functionalized QDs motivated us to use this material as an optical biosensor for detecting H₂O₂ and glucose molecules. MoS₂ QDs exhibit peroxidase-like activity and thus catalyze the decomposition of peroxides into OH radicals,²⁹ as shown in Fig. 4(a). The enzymatic non-mediator based process requires a proximal distance between MoS₂ QDs (sensor) and H₂O₂ (analyte), allowing it to be interpreted by the FRET process, involving molecular interaction between the donor and acceptor.³⁰ The FRET process was examined experimentally by measuring the quenching of the PL intensity of MoS₂ QDs with increasing concentration of H₂O₂, as shown in Fig. 4(b). The surface of the MoS₂ QDs was bio-functionalized during the biosynthesis process to bring electrostatic interactions with H₂O₂ and achieve a strong donor–acceptor complex formation. The electrostatic attraction was created by the surface charge difference between MoS₂ QDs and H₂O₂, which would have resulted in a high bimolecular quenching constant and confirms a strong ground state interaction between the donor and acceptor QDs. To confirm the electrostatic interaction, we have measured the zeta potential of MoS₂ QDs after and before the addition of H₂O₂, at pH 7. We have found that the value of zeta potential reduced from −24 mV to −15.6 mV after the addition of 10 nM H₂O₂. It was further validated by the spectral overlap between the absorption spectrum of the analyte i.e. energy acceptor (H₂O₂) and emission spectrum of the sensor i.e. the energy donor (MoS₂ QDs) (Fig. S2(a)†). We have also calculated the overlap integral value (for more details, see ESI†) for H₂O₂ to be 2.40 × 10⁹ M⁻¹ cm⁻¹ nm⁴. As depicted in Fig. 4(c), there was a reduction in the PL intensity, upon increasing H₂O₂ concentration. This can be attributed to the change of MoS₂ fluorescence caused by H₂O₂ molecules and energy transfer from MoS₂ to H₂O₂.

Fig. 4 (a) Schematic of H₂O₂ detection by MoS₂ QDs, (b) PL emission spectra quenching of MoS₂ QDs in the presence of H₂O₂, (c) changes in PL spectra in the presence of H₂O₂, (d) Stern–Volmer curve fitting of MoS₂ QDs, and (e) binding constant calculation of MoS₂ QDs in the presence of H₂O₂.

To further support the binding of H₂O₂ with MoS₂ QDs, UV-Vis absorption spectra for H₂O₂ suspensions in MoS₂ QDs with concentrations of 10, 20, and 50 nM were collected. The prominent shift in the UV-Vis absorption peaks was noted upon decreasing H₂O₂ concentration (Fig. S2(b)†). The blue-shift and apparent decrease in the absorption peak confirmed the binding of H₂O₂ on the surface of MoS₂ QDs or its breaking into OH˙ radicals. The capability of the analytical system for H₂O₂ sensing was evaluated by varying the concentration of H₂O₂ in the MOS₂ QD suspension. The relationship between the H₂O₂ concentration and PL intensity indicated a linear relationship in the concentration with two linear ranges 0 to 30 nM and 30 nM to 50 nM, as shown in Fig. 4(d). The PL quenching of MOS₂ QDs with H₂O₂ was investigated using the Stern–Volmer (SV) plot graphically representing the equation

where F and F₀ refer to the fluorescence intensity estimated with and without H₂O₂ respectively, C is the concentration of H₂O₂ and K_SV represents the Stern–Volmer quenching constant. The system (MoS₂ QDs) was found to follow a linear Stern–Volmer relation, implying a static quenching mechanism. The data could be fitted using the Stern–Volmer equation with a detection limit of 43 pM, a correlation coefficient of 0.97192, and a Stern–Volmer quenching constant of 0.02167 nM⁻¹ in the 0 to 30 nM linear range. Other fitted data are shown in the linear range 30 to 50 nM with a detection limit of 87 pM, correlation coefficient of 0.95706, and Stern–Volmer quenching constant of 0.65971 nM⁻¹ in Table S3.† The detection limit was found to be superior to that of other sensing methods (given in Fig. S4†).^31–33 The detection limit was estimated by using the equationwhere σ is the standard deviation of blank MoS₂ QDs and samples recorded (n = 5) and S is the slope of the calibration plot. The binding constant of the H₂O₂ with MoS₂ QDs was 0.0257 nM⁻¹, which was calculated by the Benesi–Hildebrand method, as shown in Fig. 4(e). The surface groups on MoS₂ QDs as depicted by the FTIR study are exploited for their affinity for glucose and studied for its detection.

Ordinarily, glucose detection by QDs requires the enzyme conjugation, where QDs couple with glucose oxidase to break down glucose molecules into gluconic acid and H₂O₂.³⁴ For the present method, owing to the favorable synthesis protocols of the biocompatible QDs, glucose molecules could bind to the surface groups present on the QDs (from the FTIR spectrum given in Fig. 2(c)). The glucose has been hypothesized to bind to either surface hydroxyl or nitro groups or the glucose binding proteins present on MoS₂ QDs, as shown in Fig. 5(a). The overlap between the absorption spectrum of glucose and the PL emission spectrum of MoS₂ QDs confirmed the FRET between glucose and MoS₂ QDs (Fig. S3(a)†). We have also calculated the overlap integral (for more details, see ESI†) values for glucose to be 4.11 × 10⁹ M⁻¹ cm⁻¹ nm⁴. The PL emission intensity of MoS₂ QDs was found to decrease as a result of glucose addition, and the change in the PL emission intensity is shown in Fig. 5(b) and (c). A change in the UV Vis spectra of glucose, with a characteristic peak at 270 nm, was observed upon MoS₂ QD addition with different concentrations (Fig. S3(b)†). There was an apparent decrease in the absorption peak, which validated glucose binding or complex formation with MoS₂ surface bonds. Additionally, there was a slight increment in the PL intensity up to a glucose concentration of 30 nM, which was followed by an exponential rise until 50 nM (Fig. 5(d)). This can only happen if both static and dynamic PL quenching occur simultaneously.

Fig. 5 (a) Schematic of glucose detection by MoS₂ QDs, (b) PL emission spectra quenching of MoS₂ QDs in the presence of glucose, (c) changes in PL spectra in the presence of glucose, (d) Stern–Volmer curve fitting of MoS₂ QDs, and (e) binding constant calculation of MoS₂ QDs in the presence of glucose.

The PL emission intensity of glucose with MoS₂ QDs was also studied using the SV equation. This indicated two linear behaviors. The data could be fitted using the SV equation with a detection limit of 2 pM, a correlation coefficient of 0.99849, and a Stern–Volmer quenching constant of 0.02352 nM⁻¹ in the 0 to 30 nM linear range. Other fitted data shown a linear range 30 to 50 nM with a detection limit of 68 pM, a correlation coefficient of 0.94582, and a Stern–Volmer quenching constant of 0.00710 nM⁻¹ (Fig. 5(d)), which makes the sensing system highly sensitive as compared to previously used methods(Table S4 and Fig. S4(b)†).^35–37 The binding constant of the glucose with MoS₂ QDs was 0.00566 nM⁻¹, which was calculated by the Benesi–Hildebrand method, as shown in Fig. 5(e).

The FRET efficiency (E) of the system for H₂O₂ and glucose sensing by MoS₂ QDs was calculated using the equation given below

where E is the efficiency and F₀ and F are the PL emission intensities recorded in the absence and presence of glucose and H₂O₂ respectively. In the case of H₂O₂, consistent FRET efficiency i.e. ∼20% was estimated for a concentration from 30 to 50 nM while for glucose, the most efficient energy transfer was observed to be 23% only at 50 nM. The FRET efficiency comparison for H₂O₂ and glucose for varying concentration is shown in Fig. S5.† From the obtained results, MoS₂ QDs were established as the potential candidate for sensing analytes, which can be extended to other similar biomolecules. The selectivity of the as-prepared QDs towards the H₂O₂ and glucose was confirmed (see Fig. S6(a)†) along with an estimation of the effective pH for the detection of the molecules. The QDs have been found to be highly selective towards the substrates under study i.e. glucose and H₂O₂ (Fig. S6(b)†)working efficiently at a pH value of 7. The recovery percentage for the substrates was calculated from Table S4.†

The stability of the microbially produced bio-conjugated MoS₂ QDs was examined by studying their QY for two months i.e. 60 days (see Fig. S7(b)†) with a comparative plot demonstrating the QY (%) for different methods of MoS₂ QD preparation^38–42 (Fig. S7(a)†). The stability of the biologically prepared QDs can be attributed to the presence of protein molecules in the growth media which prevent their aggregation and allow them to stay stable for an effective period.⁴³

4. Conclusions

In summary, we developed a strategy to fabricate surface-functionalized MoS₂ QDs utilizing a sulphide producing facultative anaerobe Pseudomonas aeruginosa. Pseudomonas aeruginosa was selected from a set of cysteine metabolizing bacteria with diverse functions and found to be affirmatively involved in the biological production of MoS₂ QDs. This is attributed to changes in pathways involved in cysteine metabolism in P. aeruginosa in comparison to others. The organic surface bonds on MoS₂ QDs, detected by FTIR, are impacted by media and bacterial growth components. Similarly, the optical property of the QDs was also influenced by the precursor concentration and cell growth phase. The surface organic groups reinforced the ultra-low detection of glucose and H₂O₂via promoting the reaction affinity between the QDs and analytes. From the results, few-layered approximately 6 nm MoS₂ QDs with a bandgap of ∼2 eV and a quantum yield of 40% achieved a LOD value of 2 pM and 43 pM in the linear range 0–30 nM for glucose and H₂O₂ respectively. The amenability of the method allows the tailoring of the process to engineer new materials for advanced applications in biological and medicinal areas.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors acknowledge the Central Research Facility of the Indian Institute of Technology, Kharagpur, for providing high-end instruments required in the sample analysis.

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: 10.1039/d0bm01206h

‡ These authors contributed equally.

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