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Detection of Mucin using Smartphone Based Platform

Chapter 5: Phenylboronic Acid Templated Gold Nanoclusters for Mucin Detection Using a

5.7. Detection of Mucin using Smartphone Based Platform

TARGETED CANCER CELL THERANOSTICS

Figure D5.11d). Confocal images of control HeLa spheroids without PB-Au NC treatment did not exhibit any fluorescence (Appendix D, Figure D5.12a-c). For evaluation of the therapeutic potential, the spheroids were treated with different concentrations of PB-Au NCs and were analyzed by AO–EtBr (live and dead) assay to identify living (green) and dead (red) cells. At 6 mg/mL PB- Au NCs, the bright-field images showed disintegration of the tumor spheres and the amount of dead cells (represented by red in the double-staining assay) was also found to be maximum at this dose (Figure 5.6A-H). These results indicated the successful application of a PB-Au NC probe for labeling and therapeutic response toward multicellular spheroids.

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concentrations of mucin recorded with a fluorescence spectrophotometer and a smartphone-based device. The values are represented as the mean ± standard deviation of three individual experiments. (D) Correlation of fluorescence measurement between a fluorescence spectrophotometer and a smartphone-based device.

Interestingly, when the PB-Au NCs were allowed to interact with mucin type III from porcine stomach (examined as a model system for human mucin glycoprotein), it was observed that their interaction could enhance the luminescence intensity. Upon the addition of increasing amounts of mucin to the PB-Au NC probe, the luminescence increased proportionately (Appendix D, Figure D5.14a). Also, the increase in the mucin concentration could be detected in terms of the luminescence enhancement of PB-Au NCs when measured in the presence of FBS spiked with mucin (Appendix D, Figure D5.15a). Subsequently, the detection of mucin was also carried out in human plasma. The increase in the mucin concentration in terms of the luminescence enhancement was observed in human plasma spiked with mucin. This indicates the potential of using the probe in clinical samples (Appendix D, Figure D5.15b). The stability of the PB-Au NC probe was studied in human plasma for up to 24 h. The results revealed the stability of PB-Au NCs in human plasma, and no loss of luminescence was observed for up to 24 h (Appendix D, Figure D5.15c). In addition, hemolysis assay indicated the blood compatibility of the PB-Au NC probe because no significant percentage of hemolysis was observed (Appendix D, Figure D5.15d). For ascertaining the specificity of these PB- Au NCs toward mucin, other possible interfering analytes such as glucose, trypsin, lipase, HSA, and α-amyloglucosidase were tested. The luminescence intensity of PB-Au NCs was found to specifically increase with respect to mucin (Appendix D, Figure D5.14b.). This behavior was possibly due to the interaction between sialic acid moieties on the mucin surface, with the boronic acid template leading to a favorable reduction in the intraparticular distance between Au NCs, causing an enhanced luminescence effect. This was supported by OD measurements where the interaction between mucin and PB-Au NCs(31) marked an increase in the turbidity of the PB-Au NC suspension in comparison to other controls (Appendix D, Figure D5.15e). Also, the TEM image after the addition of mucin revealed the presence of aggregation. The possibility of electrostatic interaction was negligible because the ζ potentials of both PB-Au NCs and mucin at physiological pH were negative (−6.1 ± 0.16 and −18 ± 1.4, respectively; Figure D5.16a-d).

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A

B

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Figure 5.8. (A-C) Snapshots of the work flow of the custom designed application.

Upon identification that the simple addition of mucin on the synthesized PB-Au NCs would allow its immediate and specific detection as a virtue of an increase in the luminescence signal, we worked toward the development of a POC platform based on a mobile phone to integrate this assay for the rapid estimation of mucin using the luminescence of Au NCs. Here, the system offers two advantages that are of primary interest for the development of POC assays. First, the synthesis of Au NCs on PB is rapid, involving a simple procedure. Second, changes in the luminescence can be achieved immediately after the addition of mucin. These significantly reduce the time and being a “one-step”

procedure requires no additional functionalization steps. The compact device consists of an a UV- LED (300 nm) for excitation of the sample in a custom-designed cuvette. The device has a height- adjustable platform to mount the mobile phone at the desired working distance, and its camera is used to acquire images of the emitted light from the sample (in a perpendicular direction to the excitation light source) through the emission filter and lens. A user-friendly software application was developed to obtain intensities of the emitted light and estimate the concentration of the samples through image analysis (Figure 5.7A). Parts A–C of Figure 5.8 shows screen shots of the application developed for performing the fluorometric quantification and the detailed approach followed in this process. To employ the device for mucin quantification, calibration was initially carried out by

C

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acquiring images of luminescent samples of PB-Au NCs after interacting with known concentrations of mucin and analyzing the image intensities by the custom-designed software application. It was found that the obtained intensities and concentrations of mucin (in the range of 0–1000 μg/mL) were to be best fit with a second-order polynomial. The obtained relation was then compared to that obtained by the standard spectrofluorometer to evaluate its performance. The fluorescence intensity from the standard spectrofluorometer and the concentration of mucin were also found to best fit with a second-order polynomial similar to that obtained by the device. A good correlation was observed between fluorescence measurements with the spectrofluorometer and smartphone-based device with a correlation coefficient (r) equal to 0.98. On the basis of the experiments, the detection limit was found to be 25 μg/mL. Alternatively, using the obtained second-order relationships, the limit of detection determined by adding 3 times the standard deviation to the mean of the control sample (with no mucin) was found to be 42.8 μg/mL for the device and 42.7 μg/mL for the standard fluorescence spectrophotometer, which is suitable for detecting mucin concentrations present in body fluids(32-35) (Figure 5.7B–D). These results demonstrate that the performance of the POC device toward mucin detection is comparable to those of standard fluorescence spectrophotometric techniques. To the best of our knowledge, the time taken for probe development and mucin detection in the present work is the shortest among previously reported methods, which enables it to be a rapid POC assay. In this context, the earlier works report time-consuming probe development techniques, functionalization, and, thereafter, long incubation periods on the sensor surface for reactions to occur.(36)