Bacterial contamination of water and food is a grave health concern rendering humans quite vulnerable to disease(s), and proving, at times, fatal too. Exploration of the novel diagnostic tools is, accordingly, highly called for to ensure rapid detection of the pathogenic bacteria, particularly Escherichia coli. The current manuscript, accordingly, reports the use of silane-functionalized glass matrices and antibody-conjugated cadmium telluride (CdTe) quantum dots (QDs) for efficient detection of E. coli. Synthesis of QDs (size: 5.4–6.8 nm) using mercaptopropionic acid (MPA) stabilizer yielded stable photoluminescence (∼62%), corroborating superior fluorescent characteristics. A test sample, when added to antibody-conjugated matrices, followed by antibody-conjugated CdTe-MPA QDs, formed a pathogen-antibody QDs complex. The latter, during confocal microscopy, demonstrated rapid detection of the selectively captured pathogenic bacteria (10 microorganism cells/10 μL) with enhanced sensitivity and specificity. The work, overall, encompasses establishment and design of an innovative detection platform in microbial diagnostics for rapid capturing of pathogens in water and food samples.

  • Antibody-mediated fluorescent biosensor for efficient capturing and detection of E. coli.

  • Bioconjugation of QDs and E. coli antibodies.

  • Qualitative, quantitative and selective assessment of E. coli in contaminated water using CLSM.

  • High sensitivity of glass matrix biosensor, with detection limit of 10 microorganisms/10 mL.

  • Capability of technology extension for detecting other microbial pathogens.

Graphical Abstract

Graphical Abstract
Graphical Abstract

Hygienic food and water are the primary requirements for human survival. Nearly 60% of annual deaths are reported to occur owing to water-borne diseases alone (Lee et al. 2018). Biological pathogens, as plausible contaminants in water, constitute one of the major factors accounting for mortality among humans, the presence of which in potable water has been attributed to lack of sewage management and apt sanitation facilities (Alahi & Mukhopadhyay 2017). The primary bacterial pathogens, in this regard, encompass Escherichia coli, Leptospira interrogans, Salmonella enterica, Campylobacter jejuni, Staphylococcus aureus, Clostridium difficile, Listeria monocytogenes, Bacillus cereus and Vibrio cholera (Bhardwaj et al. 2019). Among these, E. coli, a Gram-negative bacterium, is abundantly found in the lower intestine of humans. Strains of E. coli are invariably pathogenic, causing gastro-intestinal diseases, urinary tract infections and even bacterial meningitis in the neonates. Being considered as a zero-tolerance pathogen, there is an ardent need for novel and efficient approaches for rapid diagnosis, and subsequent prevention and therapeutic management of E. coli infections. Traditional approaches employed in recent years, such as culturing of bacteria on agar plates followed by standard biochemical identifications (Law et al. 2015), ELISA test involving multiple washing steps (Pang et al. 2018; Wu et al. 2019), flow cytometry, 24-h pre-treatment procedure in lateral flow assay (Luo et al. 2020), polymerase chain reaction, immune-magnetic separation and bacteria-specific aptamers with microchip capillary electrophoresis-coupled laser-induced fluorescence (Zhang et al. 2019) have resulted in different degrees of fruition, but were found to quite time-consuming, more laborious, less sensitive, expensive and requiring skilled technical manpower (Bursle & Robson 2016). Nanotechnology has provided considerable opportunities to address these problems by closely integrating diagnosis with therapeutic management of various ailments. Nanoparticle (NP)-based molecular imaging and therapy, therefore, have been successfully explored as a powerful model for non-invasive disease diagnosis (Liu et al. 2007; Nie et al. 2007).

The use of quantum dots (QDs) in biosensor diagnostic devices has undertaken for designing various devices to produce signals proportional to the concentration of the desired analyte and serve as an efficient detection tool (Mansuriya & Altintas 2020; Li et al. 2021). These are the semiconducting nanocrystals or NPs that demonstrate properties ranging between those of discrete molecules and bulk semiconductors (Grabolle et al. 2009; Kuzyniak et al. 2014). QDs offer stellar advantages over the fluorescent dyes, like intense and stable fluorescence for longer times, and resistance to photo-bleaching. Because of their minuscule size, high surface-to-volume ratio and high sensitivity, these QDs have proved to be efficient detection tools (Acharya et al. 2017; Bedi et al. 2022; Pandit et al. 2022; Petronella et al. 2022). Some recent studies have been reported, employing antibody QDs for successful detection of the pathogens in various foods (Du et al. 2021).

The present research work, therefore, endeavors to explore a novel, cost-effective, quick, specific and sensitive technique for detecting the presence of the pathogens in water samples. The work focuses on the development of bioconjugated cadmium-based QDs with significant fluorescent properties for efficient detection of pathogenic bacterium, i.e., E. coli ATCC 25922, adopting a sandwich technique employing antibody-conjugated QDs on layers of bacteria, immobilized with silane-functionalized glass matrices. The novelty of our work is to develop a glass matrices-based biosensor employing the use of antibodies as a biorecognition target which will be able to capture the pathogenic bacteria in aqueous sample and in turn produce a transducing signal in lieu of fluorescence produced by the cadmium-based QDs. A provisional patent on the current work, successfully filed by us in 2019, has very recently been published too. Subsequently, taking ostensible leads from our patent (Singh et al. 2020), another study, employing such as sandwich technique and cadmium-based QDs, has recently been reported (Ravikumar et al. 2020). Accordingly, the authors herein report the explicit details of our research work, carried out in a systematic manner.

Materials

Cadmium perchlorate (Sigma-Aldrich, St. Louis, MO, USA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimidehydrochloride (EDC) (TCI, Tokyo, Japan), 1-octadecene, (3-aminopropyl) triethoxysilane, (3-aminopropyl) triethoxysilane (APTES) and 3-mercaptopropionic acid (3-MPA) were purchased from M/s Alfa Aesar, Heysham, England, 1,2-dichlorobenzene (LobaChemie, Pvt. Ltd Mumbai, India), zinc telluride (ZnTe) (M/s Fisher Scientific, Mumbai, India), E. coli ATCC 25922 and Goat anti-E. coli antibodies were purchased from M/s Bio-Rad Labs, Gurugram, India. Anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) monoclonal antibody and goat anti-mouse IgG secondary antibody-HRP (horse radish peroxidase) conjugate were purchased from M/s Invitrogen, Thermo Fisher Scientific, Bangalore, India, Bovine serum albumin (BSA) from M/S Sigma-Aldrich, St. Louis, MO, USA.

Methods

Synthesis of cadmium telluride-based (CdTe) QDs by direct aqueous synthesis

CdTe QDs were synthesized employing a direct aqueous method, using thiol stabilizer (Singh et al. 2016). The overall scheme for synthesis is depicted in Supplementary Figure S5.

Bioconjugation of QDs

Bioconjugation of QDs with Bovine serum albumin (BSA)

CdTe QDs (1 mL, 31 nmol mL−1) were activated by adding 25 μL of N′-ethylcarbodiimide hydrochloride (EDC; 400 mM in methanol) and 25 μL of N-hydroxysuccinimide (NHS; 100 mM in methanol) under constant stirring at room temperature (RT) for 30 min. Subsequently, the activated CdTe-MPA QDs were mixed with 10 μL of 1 and 2 mg mL−1 of BSA in distilled water, and allowed to react for 2 h at RT. BSA-conjugated QDs were thus separated as a pellet from the excess of free BSA by centrifugation at 5,000 rpm (4,555g) for 15 min. This was repeated thrice for complete removal of free BSA, and the purified BSA-conjugated QDs were stored at 4 °C, followed by a process reported previously (Donegan & Rakovich 2016; Shamsipour et al. 2019). The schematic flowchart in Supplementary Figure S6(a) illustrates the preparation of bioconjugation of QDs.

Bioconjugation of QDs with antibodies

CdTe-MPA QDs were activated using EDC and NHS, as described earlier. Activated QDs were mixed with 10 μg mL−1 of goat anti-E. coli antibodies in PBS (pH 7.4) and were allowed to react for 2 h at 8–10 °C. Goat anti-E. coli-conjugated QDs were thus separated thrice as sediment to remove excess goat anti-E. coli antibodies by centrifugation (5,000 rpm (4,555g), for 15 min) (Zhu et al. 2012). The purified antibody-conjugated QDs were stored at 4 °C. The schematic representation of the preparation of antibody-conjugated QDs is shown in Supplementary Figure S6(b).

Characterization of CdTe-MPA QDs

Determination of concentration
The concentration of CdTe QDs was determined using an ultraviolet–visible (UV–Vis) spectrophotometer (Perkin Elmer UV/VIS Spectrometer Lambda 35, Singapore). Absorption spectra were recorded and concentration of CdTe QDs was calculated employing Beer-Lambert's law (Equation (1)), as reported previously (Bunkoed & Kanatharana 2015).
formula
(1)
where A is the first exciton absorbance for a given sample, C is the molar concentration (mol/L) of the QDs in the sample and L is the path length (cm) of the radiation beam passing through the sample. Herein, L was fixed at 1 cm and Є is the extinction coefficient per mole of QDs (L/mol cm). The latter extinction coefficient (Є) of CdTe QDs was computed (Yu et al. 2003), using Equation (2).
formula
(2)
Absorption and fluorescence spectroscopy

The optical characteristics of the synthesized CdTe-MPA QDs were explored employing UV–Vis spectroscopy and photoluminescence (PL, Hitachi F-2500 Fluorescence Spectrophotometer, Tokyo, Japan), with PL spectra recorded at an excitation wavelength of 400 nm (Tirado-Guizar et al. 2015).

Effect of concentration on emission intensity

PL spectroscopy was employed to study the effect of concentration on the emission intensity of QDs. Different concentrations of CdTe-MPA QDs were taken and the standard plot was made by plotting these concentrations against PL intensity.

Transmission electron microscopy (TEM)

To investigate the morphology and size of the prepared CdTe-MPA QDs, TEM was carried out employing a JEOL, USA, JEM-2100 electron microscope with an acceleration voltage of 200 kV at a magnification of 230,000×. Samples were prepared for TEM analysis by depositing QDs onto the carbon-coated copper grids, followed by drying at ambient temperature. The mean of 10–20 field views of QDs was considered for recording the observations (El-Nahass et al. 2014).

Powder X-ray diffraction pattern (PXRD)

About 10–20 mg of lyophilized CdTe-MPA QDs were subsequently analyzed at 2θ between 5° and 50° with an automated diffractometer, X'Pert PRO (M/s PAN analytical BV, Almelo, Netherlands). The radiation source was produced using Cu (PW3050/60, 1.54 Å) with an applied voltage of 45 kV, and a current of 40 mA. The overlaid diffractograms were prepared using Origin Pro 8 software (Yi & Wei 2017).

Absorption and fluorescence spectroscopy of BSA-conjugated QDs

The optical properties of BSA-conjugated CdTe-MPA QDs were investigated by UV–Vis spectroscopy and PL spectroscopy as discussed in the previous section.

Measurement of the quantum yield
The quantum yield of the hydrophilic QDs was computed using Rhodamine 6G, as per a previously reported procedure (Grabolle et al. 2009). Rhodamine 6G was dissolved in absolute ethanol as the reference standard (quantum yield 100%, concentration 31 nmol mL−1) (Virzbickas et al. 2017). The relative quantum yield of the QDs was calculated using Equation (3).
formula
(3)
Atomic force microscopy (AFM)

Atomic force microscopy (AFM, Multimode 8, Bruker, Germany) was carried out to investigate the appearance, monodispersity and conjugation of QDs with BSA. An aliquot of 10 μL sample solution of BSA-conjugated QDs and non-conjugated QDs each was dropped onto silicon wafers and allowed to dry for about 2 h and then subjected to analysis (Poderys et al. 2011).

Selective capturing and detection of bacteria using glass matrices

Amine functionalization of glass matrices

Glass slides were cut into square pieces (25 × 25 mm) and sonicated using acetone for 10 min in order to clean them. After drying for 10 min, these were treated with freshly prepared Piranha solution (70% H2SO4, 30% H2O2) at 55 °C for 30 min. Piranha-treated glass matrices were then again cleaned with water, methanol, methanol–toluene mixture (1:1 v/v) and toluene, under sonication for 10 min each. Silanization was then conducted by immersing these piranha-treated glass slides in 3-aminopropyl triethoxysilane solution (APTES; 2% in toluene) at RT with mild shaking for 24 h. Subsequently, for cleaning the glass slides again, these were sonicated using toluene, methanol–toluene, 1:1 (v/v) and methanol, each for 10 min, followed by baking the treated slides at 110 °C for 1 h and stored at RT (Marques et al. 2013). Supplementary Figure S7(a) schematically represents the piranha solution treatment, followed by silanization.

Characterization of amine-functionalized glass matrices
Contact angle measurements

Piranha- and silane-treated glass matrices were subjected to contact angle measurement (KRUSS drop shape analyzer) for confirmation of surface modification. A volume of 20 μL of the water drop was used for contact angle measurement. Angle (θ) formed by a drop on the surface of the matrix was recorded using a high-speed framing camera, and images processed and stored using a computer.

Evaluation of bioconjugation of antibodies with glass matrices

Bioconjugation of antibodies to the glass matrices was evaluated using an ELISA technique, employing Anti-Glyceraldehyde-3-phosphate dehydrogenase (GADPH) monoclonal primary antibody and goat anti-mouse IgH secondary antibody-conjugated with HRP. Glass matrices with functional –NH2 groups were coupled with Anti-GADPH monoclonal antibody via glutaraldehyde chemistry. Anti-GADPH monoclonal antibody-conjugated glass matrices were added to microtitre plate wells, and washed in hexaplicate with PBS 7.4 to remove the free antibodies. Glass matrices were added to a 96-well plate along with 5% skimmed milk to block active sites for preventing non-specific binding. After blocking, it was washed with PBS pH 7.4, and goat anti-mouse IgH secondary antibody-conjugated with HRP was added onto glass matrices. The plate was incubated at 37 °C, once again for 2 h, followed by washing with PBS pH 7.4 (Zhu et al. 2012; Sahoo et al. 2019). These washings were then added into a 96-well plate. Substrate (100 μL, 3,3′,5, 5′-Tetramethylbenzidine) for HRP was added to each well of microtitre as well as a 96-well plate for 10 min in dark. To stop the enzyme-substrate reaction, HCl (0.1 N) as a blocking agent was added to each well to develop yellow color (Vashist et al. 2014). Finally, the plate was read after every 15 min and washing cycles using a micro-titre plate reader at 450 nm (Bio-Rad, Model iMark, Delhi, India).

Bioconjugation of glass matrices with goat E. coli antibody

Amine-functionalized glass matrices were activated with 5% glutaraldehyde for about 2 h at RT. After activation, these glass matrices were conjugated to goat E. coli anti-antibodies at 8–10 °C for 1 h, as schematically represented in Supplementary Figure S7(b). The conjugated glass matrices were washed thrice with PBS (pH 7.4) and stored at 2–8 °C, until used (Zhu et al. 2012).

Fluorescent characteristics of goat anti-E. coli antibody-conjugated QDs

The PL intensity of the goat anti-E. coli antibody-conjugated CdTe-MPA QDs was investigated using an excitation wavelength of 400 nm.

Gram staining

Gram staining of E. coli antibody-conjugated glass matrices was performed to confirm the attachment of pathogenic bacteria with an antibody. The goat E. coli antibody-conjugated glass matrix was washed with PBS pH 7.4, followed by the addition of the pathogenic culture of bacteria. It was kept aside for 30 min and washed again thrice with PBS pH 7.4 to remove any free bacteria. Subsequently, crystal violet, iodine, alcohol and safranin were added to the bacteria-conjugated matrix and incubated for 1 min, following each addition. The matrix was washed with running water after each addition (Beveridge 2001; Harrigan & McCance 2014) and viewed under the optical microscope (Nikon Eclipse 80i, Chiyoda-Ku, Tokyo, Japan).

Detection of pathogenic bacteria

Glass matrices-conjugated with goat anti-E. coli antibodies were taken, treated with E. coli suspension containing a varied number of bacteria (10–1,000), added onto the glass surface and incubated at 8–10 °C for 1 h, followed by washing twice with PBS, pH 7.4. A concentration of 10 μg mL−1 of anti-E. coli antibody-conjugated to the QDs containing 31 nM of CdTe-MPA QDs was selected for the detection of E. coli showing adequate fluorescence. Subsequently, these goat anti-E. coli-conjugated CdTe-MPA QDs were added to the glass matrices containing E. coli cells individually and incubated at 8–10 °C for 20–30 min, followed by washing twice with PBS (pH 7.4) (Figure 2; Sahoo et al. 2019). The bioconjugated glass matrices attached with different concentrations of E. coli cells as well as QDs were subjected to confocal laser scanning microscopy (CLSM) for the detection of E. coli. An average of 50 microscopic fields was observed on each glass matrix. Approximately, 40, 35 and 5 positive fields were discerned with glass matrices containing 1,000, 100 and 10 E. coli cells, respectively. Furthermore, calibration was plotted between 100, 200 and 300 no. of E. coli cells and PL intensity.

Synthesis of CdTe-based QDs

Figure 1 portrays the photographic image of CdTe QDs showing the fluorescent effect during various stages of the synthesis of QDs demonstrating the transition of color and intensity at a fixed excitation wavelength of 400 nm.
Figure 1

Photographic image of CdTe QDs showing different emission colors during synthesis.

Figure 1

Photographic image of CdTe QDs showing different emission colors during synthesis.

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Figure 2

Effect of concentration of quantum dots on emission spectra. The corresponding inset shows a calibration curve between conc. of QDs and photoluminescence.

Figure 2

Effect of concentration of quantum dots on emission spectra. The corresponding inset shows a calibration curve between conc. of QDs and photoluminescence.

Close modal

Characterization of CdTe-MPA QDs

Determination of concentration

Table 1 illustrates the spectrophotometric absorbance at the corresponding λmax, band gap, particle size and the deduced concentration values of synthesized CdTe-MPA QDs.

Table 1

Concentration and size of various synthesized CdTe-MPA QDs batches

SampleλmaxParticle size (nm)Band Gap (eV)Concentration (mol L−1)
CdTe 1 567 3.4 2.1 31.1 × 10−6 
CdTe 2 545 3.8 2.2 31.5 × 10−6 
CdTe 3 520 3.5 2.3 31.8 × 10−6 
CdTe 4 535 3.6 2.0 31.7 × 10−6 
CdTe 5 533 3.6 2.1 31.2 × 10−6 
SampleλmaxParticle size (nm)Band Gap (eV)Concentration (mol L−1)
CdTe 1 567 3.4 2.1 31.1 × 10−6 
CdTe 2 545 3.8 2.2 31.5 × 10−6 
CdTe 3 520 3.5 2.3 31.8 × 10−6 
CdTe 4 535 3.6 2.0 31.7 × 10−6 
CdTe 5 533 3.6 2.1 31.2 × 10−6 

Dynamic light scattering (DLS) and zeta potential measurements

The effective size as well as the size distribution of CdTe-MPA QDs, when dispersed in water, are shown in Supplementary Figure S1(a, b). The QDs exhibited relatively narrower size distribution with a mean diameter of size range 5–6 nm and a zeta potential of −31.7 mV.

Absorption and fluorescence spectroscopy

The CdTe QDs, stabilized with MPA, showed an absorption maximum at 360 nm (Supplementary Figure S1(c)). The CdTe-MPA QDs, on the other hand, showed prominent emission peaks at 600 nm, when excited at 400 nm (Supplementary Figure S1(d)).

Effect of concentration on emission intensity

Figure 2 shows the prominent emission peak of hydrophilic CdTe-MPA QDs at 600 nm, when excited at 400 nm. The corresponding inset represents the calibration curve between conc. of QDs and PL having linear correlation coefficient (R) as 0.9975 (p < 0.005).

Transmission electron microscopy

Figure 3 depicts the TEM images of two diverse batches of CdTe-MPA QDs, with particle size ranging between 5.4 and 6.8 nm.
Figure 3

TEM images of two different batches of CdTe-MPA QDs.

Figure 3

TEM images of two different batches of CdTe-MPA QDs.

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Powder X-ray diffraction (PXRD)

Supplementary Figure S2 portrays the diffractogram pattern of the lyophilized CdTe-MPA powder, with 2θ peaks at 25°, 40° and 48° diffractions.

Characterization of BSA and antibody-conjugated CdTe QDs

Bioconjugation of water-dispersible CdTe-MPA QDs with BSA and goat anti-E. coli antibodies for E. coli detection was successfully conducted, and the following results were obtained.

Absorption and fluorescence spectroscopy of BSA-conjugated QDs

Absorption spectra of BSA-conjugated QDs depict a slight bump around 280 nm, as shown in Supplementary Figure S3(a). BSA-conjugated CdTe-MPA QDs demonstrated a 50–60% reduction in emission intensity at 1 mg mL−1 BSA concentration, and a 75% reduction using a concentration of 2 mg mL−1, as portrayed in Supplementary Figure S3(b). Slight bathochromic shift in peak position toward longer wavelength was observed for BSA-conjugated QDs.

Measurement of the quantum yield

Mean PL quantum yields of the CdTe-MPA QDs and conjugated QDs were found to be 45 ± 2% and 28 ± 5%, respectively.

Fourier-transform infrared spectroscopy (FTIR)

The FTIR spectra in Supplementary Figure S4(a, b) depict carbonyl group stretching vibration at 1,690.32 cm−1 in naïve QDs to peaks 1,638.91 and 1,637.80 cm−1 in BSA-conjugated QDs.

Atomic force microscopy (AFM)

Supplementary Figure S1(e) (i, ii, iii) portrays the AFM images of non-conjugated and BSA-conjugated CdTe-MPA QDs, respectively. Supplementary Table S1 shows the root-mean-square roughness (Rq) and mean roughness (Ra) values of 1.603 and 1.273, and of 0.565 and 0.386 for BSA-conjugated QDs, respectively.

Selective capturing and detection of bacteria using glass matrices

Characterization of amine-functionalized glass matrices

Contact angle measurements
Figure 4 shows the contact angle (θ) values of the water droplets as observed on various glass surfaces. The values of θ on the glass surface, after piranha solution treatment and silane functionalization, are enlisted in Table 2 indicating enhancement in water contact angle.
Table 2

Water contact angle (θ) measurement of the glass matrix

S NoGlass matrixContact angle (°) mean ± SD
Piranha solution-treated glass matrix 30.65 ± 0.63 
Silane-functionalized glass matrix 67.45 ± 2.95 
S NoGlass matrixContact angle (°) mean ± SD
Piranha solution-treated glass matrix 30.65 ± 0.63 
Silane-functionalized glass matrix 67.45 ± 2.95 
Figure 4

Water contact angle measurement (θ) on the surface of (a) piranha solution-treated glass matrix and (b) silane-functionalized glass matrix.

Figure 4

Water contact angle measurement (θ) on the surface of (a) piranha solution-treated glass matrix and (b) silane-functionalized glass matrix.

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Evaluation of bioconjugation of antibodies with glass matrices

Figure 5 shows the absorbance values of GAPDH monoclonal antibody-conjugated glass matrices and washings.
Figure 5

Values of optical density observed during ELISA for confirming bioconjugation of GADPH monoclonal antibody to amine-functionalized glass matrices. The cross-bars indicate ± 1 SD.

Figure 5

Values of optical density observed during ELISA for confirming bioconjugation of GADPH monoclonal antibody to amine-functionalized glass matrices. The cross-bars indicate ± 1 SD.

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Fluorescent properties of antibody-conjugated QDs

Figure 6 depicts the emission spectra of the non-conjugated and goat anti-E. coli antibody-conjugated CdTe-MPA QDs with a prominent emission peak at 600 and 610 nm, respectively, after excitation at 400 nm.
Figure 6

Photoluminescence spectra of non-conjugated and anti-E. coli antibody-conjugated CdTe-MPA QDs.

Figure 6

Photoluminescence spectra of non-conjugated and anti-E. coli antibody-conjugated CdTe-MPA QDs.

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Gram staining

Figure 7 exhibits pink-colored optical microscopic images of the glass slides, incubated with antibody-attached E. coli, on treatment with different chemicals.
Figure 7

Glass slides incubated with antibody-attached E. coli, as observed under the optical microscope.

Figure 7

Glass slides incubated with antibody-attached E. coli, as observed under the optical microscope.

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3.4.5. Detection of pathogenic bacteria

Intense yellow fluorescence was observed, when antibody-conjugated glass matrices containing E. coli cells were incubated with anti-E. coli antibody-conjugated QDs. Likewise, the glass matrices, when incubated with 10, 100 and 1,000 cells, showed intense yellow-colored fluorescence under the CLSM field (Figure 8). The corresponding inset represents the calibration curve between no. of bacterial cells and PL having linear correlation coefficient (R) as 0.998.
Figure 8

Confocal laser microscopic images detected with anti-E. coli antibody-conjugated CdTe QDs captured on functionalized glass matrices with (a) 1,000, (b) 100 and (c) 10 E. coli cells. The corresponding inset shows a calibration curve between no. of bacterial cells and photoluminescence.

Figure 8

Confocal laser microscopic images detected with anti-E. coli antibody-conjugated CdTe QDs captured on functionalized glass matrices with (a) 1,000, (b) 100 and (c) 10 E. coli cells. The corresponding inset shows a calibration curve between no. of bacterial cells and photoluminescence.

Close modal

Table 1 enumerates the values of λmax values of the synthesized QDs, ranging between 533 and 567 nm, band gap between 2.0 and 2.3 eV, and concentration between 31.1 and 31.8 μmol/L (Table 1), indicating the reproducibility and robustness of the synthesis protocol of QDs during our work. On increasing the reflux time during the synthesis, the fluorescence color of CdTe QDs under UV irradiation changed from green to red, i.e., toward higher wavelengths (Figure 1), ostensibly due to increased size of QDs, as a consequence of reduced quantum confinement (Smith et al. 2009; Liu et al. 2010; Shen et al. 2013). It was therefore deduced that the size of CdTe QDs is directly proportional to the emission fluorescence wavelength, which could be controlled by the reflux duration. The colloidal stability and particle size, as measured using the DLS study (Supplementary Figure S1(a, b)), depicted that the CdTe-MPA QDs were formed with a narrow size distribution and high magnitude of zeta potential, i.e., −31.7 mV, indicating the potential robustness of QDs owing to strong repulsive forces, thus preventing any possible aggregation of NPs (Kalangi et al. 2018; Li et al. 2020). The size distribution and stability were further confirmed using TEM analysis too (Figure 3), delineating the formation of monodispersed spherical-shaped QDs (Wang & Qiu 2016). The emission spectrum of CdTe-MPA QDs (Supplementary Figure S1(c)) exhibited quite symmetrical and narrow spectral width (Supplementary Figure S1(d)), indicating adequate spectral resolution for the quantitative detection of the fluorescence intensity. Increasing the concentration of QDs caused a significant rise in the emission intensity (Figure 2), with hardly any shift in emission wavelength. This can be assigned to the uniform dispersion of QDs in PBS (pH 7.4) and non-interference of ligands attached to the surface of QDs (Singh et al. 2011). The diffractogram of QDs (Figure 8) showed a broad peak at 2θ = 25°, attributable to the nanocrystalline nature of MPA-capped CdTe QDs, in consonance with previous literature (Ung et al. 2012).

BSA was selected for bioconjugation, as a high molecular weight protein possesses physicochemical properties quite analogous to that of the antibodies, which are to be ultimately employed during further studies (Singh et al. 2014). Successful bioconjugation of proteins and antibodies to carboxylated CdTe QDs could be rationally attributed to EDC, the most commonly used zero-length cross-linker, on account of its high efficiency and resultant high yield of bioconjugate formation in a controlled manner (Grabarek & Gergely 1990).

A concentration-dependent reduction in the fluorescent intensity of QDs was noticeable on bioconjugation with BSA, ascribable to the possible change in the electronic energy of QDs, owing to interactions of these ligands with the surface of QDs (Singh et al. 2015). The modified QDs, nevertheless, were found to retain substantial fluorescent properties (Supplementary Figure S3(a, b)). Furthermore, the mean quantum yield of conjugated QDs relative to that of non-conjugated ones was about 62%, suggesting substantial retention in the fluorescent characteristics of the QDs, even after bioconjugation (Ferrari & Bergquist 2007; Virzbickas et al. 2017).

The FTIR spectroscopy (Supplementary Figure S4(a, b)) was employed to investigate the optical properties of QDs, it being a versatile technique for identification and characterization of various functional groups present on the surface of QDs (Fine et al. 2020). The presence of BSA around CdTe-MPA QDs can be deciphered by the existence of all the main signals, with almost similar C = O stretching vibration of the carbonyl groups at 1,638.91 and 1,637.80 cm−1 (Singh et al. 2011). Such results formed the basis for further bioconjugation studies with goat anti-E. coli antibodies. Also, the AFM images of BSA-conjugated QDs, with Rq and Ra values of 0.565 and 0.386, respectively (Supplementary Figure S1(e) and Table S1), construed smoothness of the BSA-conjugated QDs and their potential stability, after bioconjugation with ligands (Abha et al. 2020).

Subjecting antibody-bioconjugation to EDC/NHS activation resulted in the formation of highly reactive intermediate, i.e., NHS-carboxylate, and desired nanoconjugates of the antibody with CdTe-MPA QDs (Tripathi et al. 2016). The fluorescent nature of the non-conjugated QDs and goat anti-E. coli antibody-conjugated QDs (Figure 6) depict a fourfold reduction in the emission intensity with minuscule bathochromic shift, i.e., peak position shifting toward longer wavelength. The aforesaid properties of QDs vividly indicate the presence of antibody on the surface of QDs and their vast promise as efficient fluorescent signal-producing tools for detecting microorganisms, like E. coli. Hence, successful bioconjugation of anti-E. coli antibodies with QDs was corroborated by the decline in their emission intensity vis-à-vis that of the unconjugated CdTe-MPA QDs, delineating the presence of antibody on the surface of QDs.

Since QDs need a suitable platform to efficiently capture the bacteria and assist in their detection, the glass matrices were employed for the purpose. Such glass matrices are documented to be hydrophobic, optically transparent and effective for selective capturing of pathogens after apt chemical or biological modification (Renuka et al. 2018). Hence, these were further surface-functionalized with amine groups using piranha solution with θ value of 30.65 (±0.63), as the generation of hydroxyl groups on the outer surface area of glass matrix resulted in more hydrophilic characteristics (Marques et al. 2013). Accordingly, the functionalized glass matrix was silanized (Supplementary Figure S7(a)), resulting in the augmentation of hydrophobicity and water contact angle of 67.45 ± 2.95 (Table 2), thus enabling feasible antibody conjugation. On the basis of the absorbance values of GADPH monoclonal antibody-conjugated glass matrices, determined periodically after every 15 min, and washing cycles (Figure 5) using the ELISA technique, an optimum incubation time of 2 h was selected indicating the efficiency of glass matrices for bioconjugation with the antibody (Tripathi et al. 2016; Chandan et al. 2018). After these successive washings and 2 h of incubation time, the absorbance of these functionalized glass matrices (10 μg mL−1 GADPH monoclonal antibody) tended to decrease due to the removal of free (i.e., unconjugated) antibodies. Lastly, a high absorbance value (i.e., 0.41) was observed signifying the complete removal of free antibodies and their potential for further bioconjugation with E. coli-specific antibody(ies).

Bioconjugation of anti-E. coli antibodies with glass matrices was accomplished by the formation of covalent bond via glutaraldehyde chemistry (Renuka et al. 2018; Supplementary Figure S7(a)). Gram staining using safranin clearly indicated the presence of pink color (Figure 7), ostensibly owing to the existence of a thick mesh-like cell wall, made up of peptidoglycan (50–90% of cell envelope) for a Gram-negative bacterium, i.e., E. coli (Beveridge 2001). This antigen/antibody interaction on glass matrices rendered selective capturing of E. coli cells by antibody-conjugated glass matrices, thus paving a way for developing a fluorescent-based detection probe employing QDs (Bhardwaj et al. 2017). The presence of yellow-colored fluorescence, observed during CLSM imaging (Figure 8), confirmed the attachment of E. coli antibody-conjugated glass matrices containing 1,000, 100 and 10 E. coli cells and CdTe-MPA QDs conjugated with anti-E. coli antibody. The calibration curve between no. of bacterial cells and PL illustrated a linear correlation coefficient (R) as 0.998 indicating effective capturing of 100, 200 and 300 E. coli cells, respectively, by the anti-E. coli antibody-conjugated QDs.

Overall, the CdTe QDs, synthesized using MPA-a thiol stabilizer, exhibited improved optical properties, aqueous dispersibility and uniform particle size. It may be ascribed to the tendency of the thiol molecule to firmly stick to the surface of the resultant QDs via Cd–SH bonds, thus preventing the uncontrollable nuclei growth (Li et al. 2011; Kaczmarek et al. 2016). Hence, this sandwich model corroborated the utility of CdTe-MPA QDs for the successful detection of E. coli cells, i.e., 10 organisms/mL, demonstrating high sensitivity and specificity of the developed fluorescent probe.

The current research work reports the successful development of fluorescent bioconjugated-QDs for efficient detection of E. coli ATCC 25922. Synthesized using the direct aqueous method and subsequently conjugated with anti-E. coli antibodies, the CdTe-MPA QDs were found to be monodispersed and spherically shaped. The high quantum yield of conjugated CdTe-MPA QDs with significant fluorescent characteristics ratified their utility as an effective microbial detection tool. Bioconjugation of goat anti-E. coli antibodies with glass matrices, as well as QDs, studied using ELISA and CLSM, demonstrated selective detection of E. coli. In a nutshell, the silane-functionalized glass matrices and cadmium-based QDs hold considerable potential for efficient detection of E. coli in water samples, without any interference with the complexity and interaction(s) of other bacterial species, unless they are specifically surface-modified using antigen–antibody interaction(s). The aforementioned technology can further be rationally extended and explored for the detection of other pathogenic microorganisms in myriad samples too.

The authors gratefully acknowledge the financial grants received from DST-UT (19-20)/Sanc/10/2019/1703-1710, dated 31.10.2019), Government of India. R.P.B. and G.S. appreciate the support granted by UGC, New Delhi, India, under Faculty Recharge Program.

All relevant data are included in the paper or its Supplementary Information.

The authors declare there is no conflict.

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Supplementary data