Phenylboronic acid templated gold nanoclusters for mucin detection

Dec 4, 2017 - A phenylboronic acid templated gold nanocluster probe was developed to detect biomarker mucin by non-invasive fluorescence based method ...
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Phenylboronic acid templated gold nanoclusters for mucin detection using a smartphone based device and targeted cancer cell theranostics. Deepanjalee Dutta, Sunil Kumar Sailapu, Arun Chattopadhyay, and Siddhartha Sankar Ghosh ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b13782 • Publication Date (Web): 04 Dec 2017 Downloaded from http://pubs.acs.org on December 4, 2017

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Phenylboronic acid templated gold nanoclusters for mucin detection using a smartphone based device and targeted cancer cell theranostics Deepanjalee Dutta,1 Sunil Kumar Sailapu,1 Arun Chattopadhyay*1,2 and Siddhartha Sankar Ghosh*1,3 Centre for Nanotechnology,1 Department of Chemistry 2 and Department of Biosciences and Bioengineering 3 Indian Institute of Technology Guwahati, Guwahati – 781 039; India Email ID: [email protected], [email protected] KEYWORDS: phenylboronic acid, gold nanocluster, fluorescence, point of care, smartphone device, mucin, sialic acid, anticancer

ABSTRACT: A phenylboronic acid templated gold nanocluster probe was developed to detect biomarker mucin by non-invasive fluorescence based method using a point of care smartphone based fluorescence detection device. The gold nanocluster probe is able to detect mucin specifically. The same probe was applied for in vitro targeted bio-imaging of HeLa and Hep G2 cancer cells and it demonstrated specific therapeutic effects towards cancer cells as well as multicellular tumor spheroids imparting theranostic property. The module is found to be more

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effective towards HeLa cells and pathway of cell death was established using flow cytometry based assays.

Introduction Biological molecules like protein receptors, aptamers, peptides, antigens, antibodies are widely accepted in cancer research for sensing, recognition, assay of biomarkers and to deliver therapeutic solutions.1 However, they lack stability when subjected to changes in pH, heat and are more susceptible to degradation during surface modifications, which is highly desirable to link other moieties such as fluorophores for detection purposes.1 Alternatively, use of small synthetic molecules for recognition of a specific analyte provide a suitable platform, which is by far more stable and can be easily modified for enhancing activity. Akin to biological interactions, these chemical molecules make use of their functional groups present on them and bind to the analyte with high specificity and affinity. In this regard, phenylboronic acid has been accredited towards targeting sialic acid residues with high specificity at physiological pH.2 A wealth of nanoparticle based delivery vehicles have been developed in the recent past targeting sialic acids on cancer cell surface.3-5 Overexpression of sialic acid residues or altered glycosylation play a pivotal role in the manifestation of many diseases including various cancers like prostate, liver, breast, cervical, stomach among many others. Further, hypoxic regions of solid tumors are also known to overexpress sialic acids.6,7 Hence, rapid sensing methods based on specific targeting of sialic acid could be useful to develop point-of-care (POC) diagnostic devices and is of importance in targeted delivery and therapeutics. An important secretory glycoprotein, mucin, contains sialic acid moieties and is found to be associated with pathways in cancer progression, invasion and metastasis. Also, mucins constitute a major portion of the tumor area/volume in

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many cases.8 However, rapid detection of mucin is challenging, as current standard methods, like histochemical analysis of tumor tissues through PAS/Alcain blue staining9, 10 is invasive, time consuming, and alternative approaches, such as enzyme-linked immunosorbent assays (ELISA), antibody mediated FET devices and aptamer based fluorometric assays,11-15 require expensive antibody mediated reactions and complicated labelling steps for signal generation. Thereby, development of rapid “one-step” assay for analysis of mucins present in body fluids, tumor biopsy etc., could enable for integration to POC platforms for in vitro diagnostics. A key element would be its specific recognition using labelling molecules for adequate signal generation using minimal precursors so as to keep the operation of the device simple, accurate and specific. In addition to targeting, boronic acids have also gained interest in anticancer therapy as potential proteasome inhibitors, enzyme inhibitors, boron neutron therapy agents etc., Drug testing has been carried out using boron containing drugs as new class of pharmaceuticals for various diseases including cancer.16-22 As a consequence, besides successful detection of cancer markers, boronic acids hold the potential to act as anticancer agent. However, for application of boronic acids as anticancer agent, it is essential to track its distribution inside the cells and can be achieved via tagging of the therapeutic boron based compounds with imaging probes. Therefore, an important factor to either use boronic acid template systems for development of rapid in vitro POC assays to detect mucin, or track intracellular distribution during therapeutic regime inside cells, is to achieve effective tagging with reporter molecules. In this regard, fluorometric markers are promising due to their fast response, sensitivity and hence are widely used in detection of biomolecular analytes and bioimaging. Though, fluorescence labeling is a daunting challenge with respect to small molecules like phenylboronic acid, it is mostly favored and considered as advantageous due to its applications in theranostics.

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Among fluorescence reporters, organic dyes had been a choice for decades, in fluorescence based studies but their applications in biological experiments is limited owing to their low photostability, toxicity (in certain cases) and poor solubility in water.23 Nanomaterials like quantum dots, C-dots etc., though a viable option, often require either high temperature conditions or purification steps, takes long time and related toxicity concerns arising from use of heavy metals in synthesis of quantum dots, hinders their extensive usage.24-27 Recently, noble metal nanoclusters of Au, Ag, Cu have emerged as promising candidates for sensing assays and biological applications due to their biocompatiblity, photostability, large Stokes shift.28 However, majority of available techniques involve pre-synthesis of nanomaterial and then require functionalization to achieve effective conjugation with receptors for recognition purposes. This not only introduces an extra step but also increases the possibilities of loss of the properties of the nanomaterial or the biological component involved. Instead, direct use of as synthesized luminescent nanomaterial is preferred, particularly for POC assays in order to reduce time, cost, resources and to avoid extra steps. However, such approaches to develop luminescence based assays using nanoclusters on small molecules, produced through rapid synthetic routes, to detect important cancer biomarker mucin has not been so far pursued. In addition, the application of the same luminescent probe on small molecule for specific labelling of cancer cells as well as tumor spheroids along with anticancer activity has not been explored. In this work, we present a rapid synthetic route of luminescent phenylboronic acid templated Au nanoclusters (PB-Au NCs) for application in in vitro POC diagnostics and to elucidate targeted anticancer therapeutic effects with simultaneous bioimaging. Towards diagnostics, we discovered a rapid “one-step” luminescent assay for mucin detection based on its interaction with indigenously developed PB-Au NC probe and further developed a mobile-phone

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based POC device with software for integration of the assay and readout. The specificity of the probe towards mucin resulted in enhancement of luminescence upon its addition and thereby allowed to develop a rapid assay to be integrated in POC device. In addition, for theranostic applications the same probe was applied to specifically label (imaging) cancer cells by virtue of luminescence from Au NCs, and simultaneously induce therapeutic effects towards cancer cells resulting from the contribution of boron component. The penetration and therapeutic efficacy of PB-Au NCs was also studied in case of multicellular spheroids of HeLa cells to better mimic the tumor environment. The uptake and detailed mechanism of cell death induced by PB-Au NCs was evaluated. Further, the antibacterial potential of the PB-Au NCs was explored so as to assess its potential in combating secondary bacterial infections in cancer. The overall work is illustrated in Scheme 1, which depicts the multiple applications of the as synthesized PB-Au NC probe for mucin detection using smartphone based platform, targeted bio-imaging, therapeutic activity

towards cancer cells and multicellular spheroids.

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Scheme 1. The schematic presents the rapid synthesis procedure of PB-Au NCs and their application in smartphone based mucin detection as well as targeted cancer cell imaging and therapy. MATERIALS AND METHODS Chemicals: HAuCl4 (Au, 17 wt% in dilute HCl; 99.99%, Sigma-Aldrich), phenylboronic acid (Sigma-Aldrich), mercaptopropionic acid (MPA)(Sigma-Aldrich), mucin from porcine stomach Type III, bound sialic acid 0.5-1.5 %, partially purified powder (Sigma-Aldrich), human serum albumin

(Sigma-Aldrich),

trypsin

(Sigma-Aldrich),

lipase

(Sigma-Aldrich),

alpha-

amyloglucosidase (MERCK), glucose (MERCK), Luria-Bertani (LB) broth, brain-heart infusion (BHI) media, Milli-Q grade water (> 18 MΩ cm-1, Millipore) were used without any alterations. Synthesis of PB-Au NCs: The synthesis of luminescent Au NCs on phenylboronic acid was a modification of our previously reported method.28 1 mL of 12 mg/mL phenylboronic acid solution was taken, and 10 µL of 0.11 M MPA, 30 µL of 10 mM HAuCl4 was added to it. The solution was heated to 95 ºC for 2 min and rapidly cooled down to 4 ºC. The sample was then centrifuged at 10000 rpm for 3 min and pellet was redispersed in water for further use. UV visible spectroscopy and luminescence measurements: UV-visible spectrophotometer (JASCO V-630) was used for recording the absorbance of all samples. All luminescence based measurements were carried out using Fluorescence spectrophotometer (LS55, Perkin Elmer). Smartphone/Mobile phone based system for luminescence measurements: The mobile phone-based system for luminescence measurements featured a custom designed dismountable portable mechanical case with smartphone mounting capability. The case contained a dedicated compartment to accommodate a custom designed cuvette, commercially obtained UV-LED

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source (300nm), optical filter and lens. The cuvette (1x1x1.5) cm holds liquid samples and was properly shielded in a way (expect on one side of lateral wall, required for incident light) to obstruct the passage of light. A battery powered UV-LED (310 nm) was placed facing the lateral wall of the cuvette to provide source of excitation for the Au NCs. A stage to mount the mobile phone was set perpendicular to the excitation light source and provided with levels of height adjustment from the cuvette ranging between 12 cm to 5 cm as shown Figure 1D. The emitted light passed through emission filter (cut off ~ 400 nm) and an additional lens (placed between the cuvette and camera) and imaged with the camera of the mobile phone. Software Application for luminescence measurement: To analyse the captured images, a software application has been developed using an open source software and image processing libraries which perform the tasks of image processing, calibration and estimation of concentration of the unknown samples. The working process of the application is as illustrated in the workflow (figure 1). In the first step, desired images were selected using the application images for analysis. During this step, selective cropping of the image can be done. In the second step, at least three images (the more, the better) were selected with known concentrations to establish the calibration equation needed for estimation of unknown samples. After this, estimated concentration values of the selected unknown images is displayed based on the best-fit equation. The application allows transformation and display of selected image in different colour models with histogram and intensity related data of desired channels. Scatter plots and calibration values along with fitted graph is displayed in the application with the option to export.

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Luminescence Quantification: With the handheld POC platform 24-bit images (ARGB) of the emitted luminescence from various samples were acquired using the mobile phone camera. A representative 300 x 300 pixel square area in each image was selected and converted to single Vchannel image (HSV model). Thereafter, the average intensities of the converted images were acquired. To acquire calibration values, the intensities of images (whose concentrations are known) were plotted against their concentration values. Thereafter, these values were fit to a second order polynomial equation. The equation was further used to obtain the concentration of the unknown samples based on the intensity acquired from the image of the emitted luminescence. Transmission electron microscopy (TEM): For TEM analysis, 7 µL of the synthesized samples were drop cast onto the TEM grids and air dried. The TEM grid was observed under transmission electron microscope operating at a maximum accelerating voltage of 200 keV (TEM; JEM 2100; Jeol, Peabody, MA, USA) Quantum yield measurements: The quantum yield (QY) measurement was done using reference quinine sulphate in 0.10 M H2SO4 solution. The equation used for calculation of QY is as follows

m n2 QY = QYr mr n 2r Here, ‘m’ is the slope of integrated luminescence intensity vs. absorbance plot, ‘n’ is the refractive index, and suffix ‘r’ indicates reference solution quinine sulphate. The absorbance and luminescence intensity measurement were carried out consecutively one after other using the

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same solution. The quantum yield of standard (QYr) is 0.54 and refractive index of water (solvent) is 1.33. Dynamic light scattering study: Malvern Zetasizer Nano ZS was employed for hydrodynamic diameter and zeta potential measurement. Hemolysis assay: To obtain red blood cells (RBCs), 2.0 mL of human blood was centrifuged at 3000 rpm for 10 min. The supernatant was removed and the RBCs were washed three times with phosphate-buffered saline (PBS). The RBCs were then re-suspended in PBS. 20µL of this RBC suspension was added to 80µL of PB-Au NCs (0.4mg/mL, 0.75mg/mL, 1.1mg/mL, 10mg/ mL). Water and PBS were taken as positive and negative controls respectively. The samples were incubated at 37 °C for 3 h and thereafter centrifuged at 3000 rpm for 10 min. The supernatant was collected and the absorbance was measured at 570 nm with reference as 655 nm. The percentage of hemolysis was calculated by

% hemolysis =

(Absorbance of sample − Absorbance of negative control) × 100 (Absorbance of positive control − Absorbance of negative control)

Cell Culture: HeLa (Human cervical carcinoma), HEK 293T (Human embryonic kidney), Hep G2 (Human hepatocellular carcinoma) cells for cell culture experiments were acquired from National Centre for Cell Sciences (NCCS), Pune, India. For culturing cells in 5 % CO2 humidified incubator at 37 ºC, Dulbecco’s Modified Eagle’s Medium supplemented with Lglutamine (4 mM), penicillin (50 units/mL), streptomycin (50 mg/mL, Sigma-Aldrich) and 10 % (v/v) foetal bovine serum (PAA Laboratories, Austria) was used. Confocal microscopy: For experiments in confocal microscopy, 1×105 HeLa, Hep G2, HEK cells were seeded on cover slips in 35 mm culture dishes and grown in 5% CO2 humidified

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incubator (37 ºC for 24 h). Thereafter, the cells were treated with PB-Au NCs for 4 h. The treated cells were fixed using 0.1% formaldehyde and 70% chilled ethanol. The cover slips were mounted onto glass slides and the ends were sealed. Control samples without treatment with PBAu NCs were prepared in a similar manner. The samples were observed (at excitation 405 nm) under Zeiss LSM 880 microscope. For sialic acid inhibition experiment, the cells were treated with free phenylboronic acid (7 mg/mL) prior to treatment with PB-Au NCs. MTT assay: For assessment of cell viability, 1×104 HeLa, Hep G2, HEK cells/well were seeded in 96-well plate and cultured overnight (37 ºC for 24 h) in 5% CO2 humidified incubator. The cells were treated with PB, PB-Au NCs for 24 h and thereafter MTT (3-(4, 5-dimethylthiazolyl2)-2, 5-diphenyltetrazolium bromide) assay was carried out. MTT is reduced by mitochondria in living cells into colored formazan. Thus, absorbance at 570 nm reveals the amount of formazan product, which directly relates to the number of live cells. The absorbance at 690 nm was subtracted (background interference). The % of cell viability was calculated as

% viable cells =

(A570 − A690) of treated cells × 100 (A570 − A690) of control cells

Uptake and activity in 3D multicellular spheroids: Multicellular spheroids of Hela cells were formed by seeding cells into low attachment wells of a 96 well plate. The low attachment was achieved by casting a layer of 0.8 % agarose onto wells. The spheroids were then subjected to treatment with PB-Au NCs and analysed by confocal microscopy to observe the internalization of PB-Au NCs. The AO-EtBr (Acridine orange- Ethidium bromide) double staining was carried out at different doses for analysing therapeutic ability of PB-Au NCs.

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Determination of reactive oxygen species (ROS): For ROS generation studies, HeLa cells were seeded at a density of 1×104 cells/well in 96-well plate, grown for 24 h and then treated for 3 h with PB-Au NCs. After the treatment, the cells were incubated for 10 min in 1 mM 2,7 dichlorofluoresceindiacetate (5 µL /well, DCFH-DA, Sigma-Aldrich). The media was discarded and cells were harvested, redispersed in fresh media. DCFH-DA, which is a non-fluorescent dye, converts to DCFH through hydrolysis inside live cells. The hence formed DCFH on oxidation by ROS converts to green fluorescent dichlorofluorescein (DCF). The samples were analysed using (Nikon ECLIPSE, TS100, Tokyo) epifluorescence microscope at an excitation wavelength of 488 nm for DCF fluorescence. Also, the fluorescence intensity was measured in a TECAN microplate reader. Cell Cycle Analysis: Propidium iodide based staining method was adopted for cell cycle analysis. HeLa cells at a density of 1x 105 cells/well were seeded in 6 well plates, grown and then treated with PB, PB-Au NCs for 24 h. The media and PBS were collected separately for both the treated and the control cells samples. The cells were then harvested by trypsinization and all the samples were centrifuged (650 rcf, 6 min). Following this, the cells were fixed under constant vortexing by slowly adding 1 mL of 70% chilled ethanol and stored at 4 ºC. The cells were then centrifuged, washed in ice-cold PBS and treated with RNase for 1 h at 55 ºC. Then 10 µL of PI (1 mg/mL) was added to all the samples and incubation was carried out in dark at 37ºC for 30 min. The samples were analysed in CytoFLEX flow cytometer (Beckman Coulter). PI fluorescence data were recorded for 15000 cells in each sample with the CytExpert program (Beckman Coulter) for further analysis. Caspase-3 assay: For caspase-3 assay, HeLa cells (1x 105 cells/well, 6 well plates) were grown for 24 h, followed by treatment with PB, PB-Au NCs for 24 h. Both the treated and control cells

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after trypsinization were fixed in 0.1% formaldehyde for 15 min. The samples were centrifuged at 650 rcf for 6 min and the pellet was redispersed in PBS. Thereafter, 0.5% Tween 20 was added to the samples and incubated in dark for 20 min. The cells were washed thrice with PBS, and 10 µL of PE conjugated anti-caspase-3 antibody was added. After incubation for half an hour at 37 ºC, the samples were analysed for PE fluorescence in CytoFLEX flow cytometer (Beckman Coulter). Fluorescence data for 15000 cells were recorded with the CytExpert program (Beckman Coulter) in each sample for further analysis. Antibacterial activity: For antibacterial activity test, ampicillin resistant E.coli and Staphylococcus aureus were selected as representatives of Gram negative bacteria and Gram positive bacteria. Different concentrations of PB-Au NCs were used to determine the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC). The bacteria (108 CFUs/ml) were grown in media in the presence of different concentrations of PB-Au NCs for 12 h. The lowest concentration at which there was no visual turbidity was taken as the MIC value of PB-Au NCs. The cultures that lacked turbidity were reinoculated in fresh media for determining MBC. The lowest concentration of the composite that was bactericidal was taken as MBC. The bacterial growth was monitored by measuring optical density (OD) at 595 nm using a UV–visible spectrophotometer.

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RESULTS AND DISCUSSION:

Figure 1. (A) Luminescence spectra of PB, PB-Au NCs. (B) TEM image of PB-Au NCs at 20 nm scale bar. (C) Luminescence spectra of PB-Au NCs with increasing mucin concentrations. (D) Smartphone based device and workflow of the procedure. (E-F) Response of PB-Au NCs with increasing concentrations of mucin recorded with fluorescence spectrophotometer and smartphone based device. The values are represented as mean ± SD of three individual

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experiments.

(G)

Correlation

of

fluorescence

measurement

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between

fluorescence

spectrophotometer and smartphone based device. The Au nanoclusters synthesized on phenylboronic acid appeared as a colourless suspension. The formation of Au NCs was suggested by the exhibition of luminescence with peak emission at 580 nm when excited by 300 nm light (Figure 1A). In addition, SPR signatures corresponding to Au nanoparticles were not observed indicating no possible contamination or formation of Au nanoparticles (Supporting information, Figure S1A). The fluorescence excitation spectrum of PB-Au NCs revealed excitation maxima at around 300 nm (Supporting information, Figure S1B). TEM images revealed the formation of nanoclusters with average size of 2.0 ± 0.5 nm (Figure 1B, Supporting information, S1C). The quantum yield of the Au NCs was found to be 3.1 % with respect to quinine sulphate as standard. The luminescence decrease rate in case of PB-Au NCs was found to be 0.73% per min compared to 4.0% per min in case of standard organic dye rhodamine 6G. This indicated that the hence prepared PB-Au NCs were more photostable than rhodamine 6G. The MALDI-TOF results revealed peak at 4764 (m/z) in case of PB-Au NCs, which corresponds to [Au18 (MPA11) +3Na+ -3H+]3- suggesting presence of 18 gold atoms in the core of the metal nanocluster (Supporting information, Figure S2 A-D). Interestingly, when these 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. On addition of increasing amounts of mucin to the PB-Au NCs probe, the luminescence increased proportionately (Figure 1C). Also, the increase in mucin concentration could be detected in terms of luminescence enhancement of PB-Au NCs when measured in presence of FBS (fetal bovine serum) spiked with mucin. Subsequently, the detection of mucin was also carried out in human plasma. The

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increase in mucin concentration in terms of luminescence enhancement was observed in human plasma spiked with mucin. This indicates the potential of using the probe in clinical samples (Supporting information, Figure S2E, F). The stability of the PB-Au NCs probe was studied in human plasma up to 24 h. The results revealed the stability of PB-Au NCs in human plasma and no loss of luminescence was observed up to 24 h (Supporting information, Figure S2G). In addition, hemolysis assay indicated the blood compatibility of the PB-Au NCs probe as no significant percentage of hemolysis was observed (Supporting information, Figure S2H). For ascertaining the specificity of these PB-Au NCs towards mucin, other possible interfering analytes such as glucose, trypsin, lipase, HSA, alpha-amyloglucosidase were tested. The luminescence intensity of PB-Au NCs was found to specifically increase with respect to mucin (Supporting information, Figure S1D). This behavior was possibly due to the interaction between sialic acid moieties on mucin surface with the boronic acid template leading towards favorable reduction in the intra-particular distance between Au NCs causing an enhanced luminescence effect. This was supported by optical density measurements where the interaction between mucin and PB-Au NCs

29

marked an increase in turbidity of the PB-Au NCs suspension in comparison

to other controls (Supporting information, Figure S2I). Also, the TEM image after addition of mucin revealed presence of aggregation. The possibility of electrostatic interaction was negligible as the zeta potential of both PB-Au NCs and mucin at physiological pH was negative (-6.1 ± 0.16 and -18 ± 1.4 respectively) (Supporting information, Figure S3A-D). On identifying that simple addition of mucin on the synthesized PB-Au NCs would allow its immediate and specific detection as a virtue of increase in luminescence signal, we worked towards development of a POC platform based on mobile phone to integrate this assay for rapid estimation of mucin using the luminescence of Au NCs. Here, the system offers two advantages

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that are of primary interest for development of POC assays. First, the synthesis of Au NCs on PB is rapid involving a simple procedure. Second, the changes in luminescence can be achieved immediately after 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 UV LED (300 nm) for excitation of the sample in a custom designed cuvette. The device has height adjustable platform to mount the mobile phone at desired working distance and its camera is used to acquire image 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 concentration of samples through image analysis (Figure 1D). The Figure S4 (A-C) shows the 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 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 concentration of mucin (in the range of 0 - 1000 µg mL−1) 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 spectroflurometer and the concentration of mucin were also found to best fit with second order polynomial similar to that obtained by the device. A good correlation was observed between fluorescence measurement with the spectrofluorometer and smartphone based device with a correlation coefficient (r) equal to 0.98. Based on the experiments, the detection limit was found to be 25 µg/mL. Alternatively, using the obtained second order relationships, the limit of detection (LoD) determined by adding

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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 standard fluorescence spectrophotometer, which is suitable for detecting mucin concentrations present in body fluids

30-33

(Figure 1E-G). These

results demonstrate that the performance of the POC device towards mucin detection is comparable to standard fluorescence spectrophotometric techniques. To the best of our knowledge, the time taken for probe development and mucin detection in the present work is 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.20,34

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Figure 2. (A) (i-iii) Bright field image, fluorescent image, merged image of HEK cells treated with PB-Au NCs. (B) (i-iii) Bright field image, fluorescent image, merged image of HeLa cells treated with PB-Au NCs. (C) (i-iii) Bright field image, fluorescent image, merged image of HEKPG2 cells treated with PB-Au NCs. Further, to analyze the potential of PB-Au NCs in targeting cancer cells, HEK, HeLa and Hep G2 cells were treated with PB-Au NCs. The intracellular uptake of PB-Au NCs was confirmed using confocal microscopy and flow cytometry by monitoring the luminescence of Au NCs present in PB-Au NCs. The results obtained from both of these techniques revealed that the

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uptake was found to be negligible for normal cell line HEK, maximum in case of HeLa and was closely followed by Hep G2 cells (Figure 2A-C, Supporting information, Figure S5A-C). The control cells without treatment of PB-Au NCs did not exhibit any fluorescence (Supporting information, Figure S6A-C). Depth projection of confocal microscopy images of HeLa cells showed internalization of PB-Au NCs in the cells (Supporting information, Figure S7D). To further confirm that PB-Au NCs interact with sialic acid residues on cancer cells surface, an inhibition assay was performed, where cancer cells were first reacted with PB alone to block the binding sites, and thereafter were treated with PB-Au NCs. The results revealed that in case of cells treated with free PB, the uptake was reduced. Thereby, the inhibition assay indicated that the uptake of the PB-Au NCs was mediated through interaction of PB and sialic acid residues on cancer cells surface (Supporting information, Figure S8A-C). The semi quantitative RT-PCR of sialyl transferase genes also revealed the over expression of ST3GALIII (in Hela, Hep G2) and ST6GALI (in HeLa) compared to normal cell line HEK which in turn effects the sialic acid expression on cancer cell surface (Supporting information, Figure S9).

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Figure 3. (A) Cell viability assay of HeLa, HEK, Hep G2 cells treated with PB-Au NCs. (B-D) Bright field images of control HeLa cells and HeLa cells treated with PB-Au NCs. (E-G) Fluorescent images of control HeLa cells and HeLa cells treated with PB-Au NCs exhibiting DCF fluorescence. Following uptake studies, the therapeutic potential of PB-Au NCs was assessed by MTT assay in all the three cell lines. As anticipated, the in vitro anti-proliferative activity was found to be maximum for the HeLa cells followed by Hep G2, HEK. The IC 50 dose was found to be 1.1 mg/mL for HeLa and the viability of Hep G2, HEK cells was around 54 % and 70 %, respectively at this dose. These results indicated that PB-Au NCs specifically showed therapeutic effects towards cancer cells (HeLa, Hep G2) over normal cells (HEK) (Figure 3A). In addition,

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the results of MTT assay with free PB treatment alone confirmed that the activity of PB was not significantly affected after formation of Au NCs (Supporting information, Figure S10A). For elucidating the mechanism of cell death, further experiments were carried out on HeLa cells as the therapeutic effect of PB-Au NCs was found to be maximum in this case. Generation of high levels of reactive oxygen species (ROS) have been attributed as an early marker of progression towards apoptosis in most of the cell death pathways followed by anticancer agents. In this regard the DCFH-DA based ROS assay was carried out to determine intracellular ROS levels. The results revealed higher amount of ROS generation in case of of PBAu NCs treated cells. This was indicated by the fluorescence intensity (measured at 530 nm emission for DCF fluorescence) which was found to be high in case of PB-Au NCs treated cells with respect to control cells (Supporting information, Figure S10B). This was supported by the fluorescence microscopic images of the cells treated with PB-Au NCs where the intensity was higher in comparison to control cells. The changes in morphology of the cells was also observed in the corresponding bright field images of the PB-Au NCs treated cells with respect to control cells (Figure 3B-G).

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Figure 4. Cell cycle analysis of (A) control HeLa cells, and HeLa cells (B) treated with PB, (C) treated with PB-AuNCs, where M1: sub G1, M2: G0/G1, M3: S, M4: G2/M. Caspase-3 assay of (D) control HeLa cells, and HeLa cells (E) treated with PB, (F) treated with PB-AuNCs, where M1: active caspase-3 negative, M2: active caspase-3 positive. Subsequently, to unravel the effects of PB-Au NCs treatment on the cell cycle distribution, cell cycle analysis was carried out using flow cytometry based propidium iodide assay. This provides vital information related to cell death mechanism. The population of cells in sub G1 phase, which represents the apoptotic cell population, was found to be more in case of both PB (5.83%), PB-Au NCs (7.12%) treated cells in comparison to control cells (1.98%) (Figure 4A-C). This indicated possible apoptosis mode of cell death. This was then confirmed via analysis of activation of caspase-3, which play a pivotal role in downstream of the apoptotic pathway. In the caspase-3 assay, anti-caspase antibody was used to label caspase-3 produced inside the cells during apoptosis. This assay confirmed a substantial increase in the active caspase-3 positive cells (apoptotic cells) in case of both PB and PB-Au NCs treated cells in comparison to control cells (Figure 4D-F). Hence, all the above results revealed the targeted

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theranostic potential of PB-Au NCs (delivering both imaging capabilities and therapy) and can be used for anticancer treatment regime.

Figure 5. (A-D) Bright field images of control HeLa spheroids, HeLa spheroids treated with increasing doses of PB-Au NCs (0 mg/mL, 1 mg/mL, 3 mg/mL, 6 mg/mL) for 24 h and stained with AO/EtBr. (E-H) Fluorescent images of control HeLa spheroids, HeLa spheroids treated with PB-Au NCs (0 mg/mL, 1 mg/mL, 3 mg/mL, 6 mg/mL) for 24 h and stained with AO/EtBr. In an attempt to establish the potential application of these NCs in a more realistic solid tumor environment, the penetration capacity and therapeutic activity of the PB-Au NCs was determined using 3D multicellular HeLa spheroids. The in vitro spheroid culture of the HeLa cells was established using agarose coated wells in 96 well plate, where with increase in the number of cells the sphere diameter was found to increase (Supporting information, Figure S11A-D). The spheres (before treatment with PB-Au NCs) were stained with AO live cell staining dye and depth projection of confocal images revealed the morphology of the stained 3D multicellular spheroid (Supporting information, Figure S12A, B). Thereafter, the spheres were treated with PB-Au NCs and the successful delivery of the PB-Au NCs into the spheroids, was

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essentially established using confocal microscopy (through luminescence of Au NCs) (Supporting information, Figure S13A-C). The depth projection of the confocal image exhibited uptake of PB-Au NCs inside the spheroids (Supporting information, Figure S13D). Confocal images of control HeLa spheroids without PB-Au NCs treatment did not exhibit any fluorescence (Supporting information, Figure S14A-C). For evaluation of therapeutic potential, the spheroids were treated with different concentrations of PB-Au NCs and were analyzed by AO/Et Br (live and dead) assay to identify living (green) and dead (red) cells. At 6 mg/mL of PB-Au NCs, the bright field images showed disintegration of 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 5A-H). These results indicated the successful application of PB-Au NCs probe for labelling and therapeutic response towards multicellular spheroids. One of the major causes of deaths in several cancer patients is the secondary bacterial infections.35-36 In this context, we evaluated the potential of PB-Au NCs as antibacterial agents. It was found that for gram negative antibiotic resistant E.coli, the MIC (minimum inhibitory concentration) was at 0.5 mg/mL and the MBC (minimum bactericidal concentration) was at 0.7 mg/mL. For gram positive Staphylococcus aureus, the MIC was at 0.8 mg/mL and the MBC was at 1 mg/mL. Further, growth characteristics of treated and control bacteria was monitored up to 12 h. The growth was found to be minimum for MBC and MIC in case of both gram positive and gram negative (Supporting information, Figure S15A-D). Hence, it was revealed that the PB-Au NCs could show antibacterial properties, which would potentially help in reduction of secondary bacterial infections in cancer. CONCLUSIONS: In brief, the work presented a rapid method to develop luminescent Au NCs using a small molecule phenylboronic acid for mucin detection and to achieve

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simultaneous imaging and therapy of cancer cells (theranostic applications). We demonstrated that luminescence of PB-Au NCs probe enhanced upon its interaction with mucin. This phenomenon led to develop a fast luminescent assay for mucin detection suitable for integrating to POC platforms. We further developed a cost-effective, portable POC device to detect the luminescence of PB-Au NCs probe and estimate mucin. The smartphone based POC device functions through image analysis based methods to carry detection of mucin involving a “onestep” procedure. In addition, the PB-Au NCs probe was demonstrated to possess the ability to deliver simultaneous targeted imaging and therapy of HeLa and Hep G2 cancer cells. This helped in assessment of distribution of phenylboronic acid as an anticancer agent inside cancer cells as well as multicellular spheroids. The pathway and mechanism of cell death initiated by PB-Au NCs was studied using flow cytometry based assays. Also, the potential antibacterial nature of the PB-Au NCs expands its application in combating possible secondary bacterial infections in some cancers. The above findings in case of cancer cells and multicellular spheroids, along with the blood compatibility and stability of the PB-Au NCs probe in human plasma indicates potential applicability of the PB-Au NCs for in vivo models and opens up new paradigm for future work. ASSOCIATED CONTENT Supporting Information. This material is available free of charge via the Internet at http://pubs.acs.org.” UV-visible spectra, Excitation spectra, zeta potential, software snapshots, quantum yield, photostability, MALDI, confocal microscopy, antibacterial studies. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected], [email protected]

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Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This study was supported by Department of Biotechnology and the Department of Electronics and

Information

Technology

(No.

5(9)/2012-NANO

(Vol.

II)),

Government

of

India. We thank financial support of the Department of Biotechnology, Government of India, (BT/PR13560/COE/34/44/2015) and (DBT-NER/Health/47/2015). We acknowledge assistance from CIF (IIT Guwahati), Bidyut Bikash Borah for help in device construction.

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36. Vento, S.; Cainelli, F.; Temesgen, Z. Lung Infections after Cancer Chemotherapy. The Lancet Oncology 2008, 9 (10), 982–992.

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