Biosensors and Bioelectronics 54 (2014) 7–14

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Direct detection of hyaluronidase in urine using cationic gold nanoparticles: A potential diagnostic test for bladder cancer Ahmed Ibrahim Nossier a, Sanaa Eissa b, Manal Fouad Ismail c, Mohamed Ahmed Hamdy c, Hassan Mohamed El-Said Azzazy d,n a

Biochemistry Department, Faculty of Pharmacy, Misr University for Science and Technology (MUST), 6th October City, Egypt Oncology Diagnostic Unit, Medical Biochemistry & Molecular Biology Department, Faculty of Medicine, Ain Shams University, Cairo, Egypt c Biochemistry Department, Faculty of Pharmacy, Cairo University, Giza, Egypt d Department of Chemistry & Yousef Jameel Science & Technology Research Center, The American University in Cairo, New Cairo, Egypt b

art ic l e i nf o

a b s t r a c t

Article history: Received 13 August 2013 Received in revised form 10 October 2013 Accepted 11 October 2013 Available online 31 October 2013

Hyaluronidase (HAase) was reported as a urinary marker of bladder cancer. In this study, a simple colorimetric gold nanoparticle (AuNP) assay was developed for rapid and sensitive detection of urinary HAase activity. Charge interaction between polyanionic hyaluronic acid (HA) and cationic AuNPs stabilized with cetyl trimethyl ammonium bromide (CTAB) led to formation of gold aggregates and a red to blue color shift. HAase digests HA into small fragments preventing the aggregation of cationic AuNPs. The nonspecific aggregation of AuNPs in urine samples was overcome by pre-treatment of samples with the polycationic chitosan that was able to agglomerate all negatively charged interfering moieties before performing the assay. The developed AuNP assay was compared with zymography for qualitative detection of urinary HAase activity in 40 bladder carcinoma patients, 11 benign bladder lesions patients and 15 normal individuals, the assay sensitivity was 82.5% vs. 65% for zymography, while the specificity for both assays was 96.1%. The absorption ratio, A530/A620 of the reacted AuNP solution was used to quantify the HAase activity. The best cut off value was 93.5 μU/ng protein, at which the sensitivity was 90% and the specificity was 80.8%.The developed colorimetric AuNP HAase assay is simple, inexpensive, and can aid noninvasive diagnosis of bladder cancer. & 2013 Elsevier B.V. All rights reserved.

Keywords: Hyaluronidase Hyaluronic acid Bladder Cancer Gold Nanoparticles Chitosan

1. Introduction Hyaluronidases (HAases) are a family of extracellular matrixdigesting endoglycosidases that digest hyaluronic acid (HA); a nonsulfated linear glycosaminoglycan consisting of repeating units of D-glucuronic acid and N-acetyl-D-glucosamine (Laurent and Fraser, 1992). HA is one of the most versatile macromolecules of the extracellular matrix of connective, growing and tumor tissues (Catterall, 1995; Delpech et al., 1997; Tammi et al., 2002). It actively regulates several physiological processes such as cell adhesion, migration and proliferation (Turley et al., 2002). HAases are present in normal tissues (e.g., liver, kidney, and testis) and the expression of these enzymes appears to be tissue specific (Zhu et al., 1994). Interestingly, HAase is termed as a spreading factor as it is essential for the spread of bacterial infections, toxins and venoms (Girish et al., 2004). Elevated plasma levels of HAase and HA have been reported to be associated with the presence of n Correspondence to: Department of Chemistry, The American University in Cairo, SSE, Rm # 1184, AUC Avenue, P.O. Box 74, New Cairo 11835, Egypt. Tel.: þ 20 226 152 559; faxþ 20 227 957 565. E-mail address: [email protected] (H.M.E. Azzazy).

0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.10.024

tumors. Angiogenic HA fragments generated by the enzymatic action of HAase have been detected in high levels in numerous human tumors, such as bladder cancers (Lokeshwar et al., 1997; Eissa et al., 2005) and prostate cancers (Lokeshwar et al., 2001). High urinary levels of HAase have been used for detection of highgrade bladder carcinoma (Eissa et al., 2010; Pham et al., 1997). Bladder carcinoma is among the five most common malignancies worldwide. It is the second most common tumor of the genitourinary tract and the second most common cause of death in patients with genitourinary tract malignancies (Howlader et al., 2011). Classical cytology and cystoscopy are the main methods for the diagnosis of patients with bladder cancer. Voided urine cytology has a lower sensitivity for detecting low-grade tumor making the frequent use of invasive cystoscopy necessary. The development of a sensitive noninvasive diagnostic test that may specifically detect bladder carcinoma in the early stages would improve the clinical outcomes as it would allow early treatment (Kaufman et al., 2009). Several methods have been reported for assaying HAase activity most of which depend on determining the amount of degraded HA generated by the action of HAase. Physicochemical methods such as turbidometry and viscometry (Bachtold and Gebhardt,

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1952; Balazs and Euler, 1952) require large amounts of enzymes and are relatively inaccurate. While, spectrophotometric methods such as fluorometric (Nakamura et al., 1990; Rich et al., 2012; Fudala et al., 2012) and colorimetric approaches (Bonner and Cantey, 1966; Gregory and Robert, 1997) are appropriate for high-throughput platforms for assaying enzymatic activity and screening inhibitors. However, these methods require substrate modifications with fluorescein or biotin at the free carboxyl groups of HA that may affect the activity of HAases. HA substrate gel zymography was also developed (Guntenhoner et al., 1992), but this method involves time-consuming prerequisite steps such as overnight soaking. Gold nanoparticles (AuNPs) display a distinctive phenomenon known as surface plasmon resonance (SPR) that depends on the AuNP size and the inter-particle distance (Rechberger et al., 2003). In colloidal solution this SPR is responsible for the red coloration of dispersed AuNPs with interparticle distance larger than the average particle diameter, and the blue coloration of aggregated AuNPs with interparticle distance smaller than the average particle diameter

(Jain et al., 2006).These exclusive optical properties have allowed the use of AuNPs in developing simple and rapid colorimetric assays for detection of biomarkers as proteins, peptides and nucleic acids with higher sensitivity and specificity than the existing detection methods (Radwan and Azzazy, 2009; Choi et al., 2010). Kim et al. (2009) developed a fast and simple method to assay HAase activity using cationic gold nanoparticles (cysteamine stabilized AuNPs). The assay is based on charge interaction between polyanionic HA and cationic AuNPs leading to formation of gold aggregates and a simultaneous red to blue color shift. After the enzymatic reaction of HAase, polymeric HA is degraded into small fragments which are unable to aggregate AuNPs. In comparison with other HAase assays, this method showed comparable or even better performance with no need for substrate modifications with fluorescin as in the fluorometric method (Nakamura et al., 1990) or any additional steps to provoke color changes of fragmented HA as in the colorimetric approach (Bonner and Cantey, 1966). However, this assay was not investigated for detection of HAase activity in clinical specimens probably due to non-specific

Fig. 1. The principle of cationic AuNP based colorimetric assay for direct detection of HAase activity. (a) Polyanionic polymer (HA) causes the aggregation of CTAB stabilized AuNPs with red to blue color shift. (b) At high concentration of HAase, enzyme molecules bind all HA molecules electrostatically preventing aggregation of AuNPs and the solution remains red. (c) At low concentration of HAase, it needs an enzymatic reaction time at 37 1C to allow the enzyme molecules to digest HA into small fragments and upon addition of AuNPs the solution remains red.

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Fig. 2. Scanning electron microscope images showing full aggregation of cationic AuNPs on polyanionic HA. 15 mL of 3 mg/mL HA was sufficient for aggregation of 100 mL of cationic AuNPs.

aggregation of cationic AuNPs on negatively charged interfering moieties that may be present in clinical specimens leading to false negative results. Chitosan is a linear polysaccharide consisting of arbitrarily distributed β (1-4)-linked D-glucosamine (deacetylated unit) and N-acetyl-D-glucosamine (acetylated unit). It is obtained by variable N-deacetylation of naturally occurring polysaccharide chitin; therefore chitosan is characterized according to the degree of deacetylation (Dutta et al., 2002). Chitosan is insoluble in water, organic solvents and aqueous bases but is soluble after stirring in aqueous acidic solution. Chitosan has excellent properties such as biodegradability, biocompatibility and low toxicity (Hundson and Smith, 1998). Due to its polycationic nature, chitosan molecules can agglomerate anionic moieties in solution to form precipitates; hence chitosan and its derivatives are used in wastewater treatment (Sridhari and Dutta, 2000). The aim of this study was to develop a simple AuNP-based colorimetric assay to directly detect HAase activity in clinical urine samples to detect bladder carcinoma. To prevent the non-specific binding of cationic AuNPs to anionic moieties which may be present in urine samples, chitosan was used as a sequestering agent. The HAase AuNP assay was compared with HA substrate gel zymography. The principle of our work as represented in Fig.1 is based on charge interaction between long chains polyanionic HA and cationic AuNPs. This leads to formation of gold aggregates with red to blue color shift (Fig.1a and Fig. 2). At high concentrations of HAase (which is positively charged at pH 4.3), all HA molecules are believed to be electrostatically sequestered by HAase molecules and upon addition of AuNP solution no aggregation occurs and the solution remains red in color even without incubation time at 37 1C (Fig. 1b). While at low concentration of HAase, incubation at 37 1C is required to allow HAase molecules to digest all HA into small fragments and upon addition of AuNPs the solution remains red (Fig. 1c). The developed assay did not consider only the ability of polyanionic polymer as such HA to cause aggregation of cationic AuNPs, but also the binding nature of HAase to HA and the effect of sample proteins on modulating HAase activity were considered.

2. Materials and methods 2.1. Materials Tetrachloroauric acid (99.99%, HAuCl4  3H2O), hyaluronidase (type I-S), human umbilical cord hyaluronic acid (Cat. no. H1507), bovine serum albumin (BSA), cetyl trimethyl ammonium bromide (CTAB) 98%, sodium borohydride, silver nitrate, ascorbic acid, acrylamide, bis-acrylamide, sodium dodecyl sulfate, sodium chloride, sodium formate, triton X-100 and high molecular weight chitosan were all obtained from Sigma Co. (St. Louis, MO). 2.2. Clinical samples collection This study was approved by the Medical Ethical Committee of Ain Shams University, Faculty of Medicine, and informed written consent to participate in the study was obtained from each participant (n ¼66) before enrollment. Patients were admitted to Ain Shams Hospital suffering from urological symptoms from January 2012 to December 2012. After cystoscopy, based on histopathologic examination, 40 patients were diagnosed as bladder cancer and 11 were diagnosed as benign urological lesions. A group of 15 healthy volunteers were recruited from the hospital laboratory staff as controls. Voided urine (30–60 mL) samples were collected from all individuals prior to treatment or surgery, and transported on ice to the laboratory. Each urine sample was centrifuged at 4000g and then the urinary supernatant was collected and divided into aliquots and stored at  80 1C. 2.3. Synthesis of CTAB stabilized AuNPs CTAB stabilized AuNPs were synthesized with certain modifications for the seed-mediated growth method (Jana et al., 2001; Nikoobakht and El-Sayed, 2001; Murphy and Jana, 2002). Briefly, CTAB solution (7.5 mL of 0.2 M) was mixed and stirred with 2.5 mL of 0.001 M HAuCl4. Ice-cold 0.01 M NaBH4 (0.6 mL) was added resulting in formation of a solution with brownish yellow color.

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The seed solution was vigorously stirred for 2 min, and then the solution was kept at 25 1C. CTAB solution (50 mL of 0.2 M) was added to 50 mL of 0.001 M HAuCl4 and the solution changed color from yellow (HAuCl4 dissolved in water) to orange (HAuCl4 dissolved in CTAB). AgNO3 solution (1 mL of 0.004 M) was added with gentle mixing. Then 700 μL of 0.0788 M ascorbic acid was added to reduce the Au3 þ ions to Au þ and the solution changed from orange to colorless (Au þ color). To this solution, 80 μL of the seed solution was added to the center of the solution, so that the seeds started to grow. 2.4. Characterization of AuNPs Size and distribution of the prepared AuNPs were characterized using field emission scanning electron microscopy (Model: Leo Supra 55, Carl Zeiss Microscopy GmbH, Germany). One drop of the AuNP solution was added onto a silicon slide that was allowed to air dry before examination. The surface charge (zeta-potential) of CTAB stabilized AuNPs was measured using a Malvern zeta-sizer (Malvern instruments Ltd, UK). The λmax for AuNPs was measured using a UV–vis spectrophotometer (Jenway 6800, Bibby scientific Ltd., UK).The absorbance of the AuNP solution was matched to 0.9 at 530 nm.

2.8. HAase activity assay by zymography The HAase activity assay was also done according to the previously described zymography method (Guntenhoner et al., 1992). Briefly, urine samples treated with chitosan (  20 mg of protein) were electrophoresed under denaturing conditions on an 8% SDS-polyacrylamide gel containing 0.17 mg/mL HA. Next, the electrophoresed proteins were renatured by washing the gel with a 3% solution of Triton X-100 for 1 h, and then the gel was incubated in a HAase assay buffer for enzymatic digestion at 37 1C for 16–18 h. Following incubation, the gel was stained sequentially with solutions of 1% Alcian blue (overnight soaking) and 0.15% Coomassie blue (for 3 h), and destained with 10% methanol /10% acetic acid solution. The presence of HAase was detected from the unstained clear band(s) in the gel.

3. Results 3.1. Synthesis and characterization of the prepared AuNPs

To optimize the HA concentration in the assay, 15 mL of serial dilutions of HA in HAase assay buffer (0.1 M sodium formate and 0.15 M NaCl, pH 4.3) was tested with 100 mL of colloidal AuNPs. The lowest concentration of HA that results in full aggregation of AuNPs was chosen.

AuNPs were prepared using the seed-mediated growth method with some modifications to produce positively charged AuNPs stabilized with CTAB. These positive charges prevent AuNPs aggregation and a red color was observed. SEM image of AuNPs and the data obtained using a zeta-sizer have revealed that the AuNPs were well dispersed as shown in Supplementary Fig.S1a. The mean diameter was found to be 46 nm (Supplementary Fig. S1b) with positive zeta-potential value 52.1 mV (Supplementary Fig. S1c). The absorption spectrum of the prepared AuNPs exhibited a single peak in the visible region with λmax at 530 nm (Fig. 4).

2.6. HAase activity assay by cationic AuNPs

3.2. Optimization of AuNP assay

The developed HAase AuNP assay was done as follows: 5 mL HAase solution of varying concentrations was added to 15 mL of 3 mg/mL HA solution in a HAase assay buffer and the mixtures were incubated at 37 1C. Several reaction incubation times (10, 30, 60, 120 min) were investigated where incubation for 60 min was found optimal (for proper identification of positive and negative specimens). At the end of incubation the enzymatic reaction was stopped by heating the mixtures in boiling water for 5 min. After cooling the mixtures to room temperature, 100 mL of colloidal AuNPs was added to each mixture and the color was observed within 1 min with the naked eye. After the addition of colloidal AuNP solution, the spectral profiles of all reaction mixtures were recorded and the ratios of absorbance at 530 and 620 nm (A530/ A620) were calculated. To study the effect of proteins on modulating HAase activity the experiment was repeated with the same enzyme concentrations but after adjusting the final protein concentration to 50 ng per reaction mixture using BSA.

3.2.1. pH and Ionic strength Reaction conditions, such as pH, temperature and salt concentration can affect the activity of HAase. Previous studies have shown that the optimum pH for bladder tumor-derived HAase was 4.3, whilst the optimum ionic strength was 0.15 M sodium chloride concentration (Pham et al., 1997; Lenormand et al., 2009). Accordingly, the assay was performed using HA solution of 3 mg/mL in a HAase assay buffer containing 0.15 M NaCl at pH 4.3.

2.5. Optimization of HA concentration

2.7. Cationic AuNPs assay for detection of HAase activity in clinical urine samples Protein concentration of normal urine samples spiked with HAase and voided urine samples obtained from all individuals enrolled in this study was determined using Bradford method (Bradford, 1976). Then the samples were buffered at pH 4.3 using 0.1 M sodium formate and 0.15 M sodium chloride. 200 mL of each sample was mixed properly with 30 mg of chitosan and then centrifuged at 18,000 rpm for 10 min. Urine supernatants were tested with colloidal AuNPs to assure the removal of all interfering substances (no aggregation).Then, 5 mL of urine supernatant (  50 ng of protein) was mixed with 15 mL of 3 mg/mL HA solution and the assay was done as mentioned previously.

3.2.2. Optimization of HA concentration As the assay is based on charge interaction between cationic AuNPs and the negatively charged HA, the concentration of AuNPs and HA solutions were optimized to obtain a better detection limit of HAase activity. In this study, 15 mL of 3 mg/mL HA solution was enough for aggregation of 100 mL of cationic AuNP solution with red to blue color shift that could be detected with naked eye. Scanning electron microscope images showed full aggregation of cationic AuNPs on polyanionic HA (Fig. 2). 3.2.3. Optimization of protein concentration Previous studies (Asteriou et al., 2006; Lenormand et al., 2007, 2008, 2009; Deschrevel et al., 2008) have reported the effect of HA–protein complex on modulating HAase activity. Accordingly, the concentrations of HA and proteins in reaction mixture were considered while measuring the HAase activity by the developed assay. The optimum protein concentration for this assay was 50 ng total proteins per reaction mixture for 45 ng HA. 3.3. Colorimetric AuNP assay for detecting HAase activity Data obtained have shown that at high concentrations of HAase the positive results were obtained even at zero minute incubation

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time at 37 1C. Accordingly, HAase at concentrations in the range of 0–240 U/mL were used to determine the lower detection limit of the assay at zero minute incubation time at 37 1C. Using this colorimetric method the lower detection limit was found to be 24 U/mL (Supplementary Fig. S2). To determine the lower detection limit of the assay after 1 h incubation time at 37 1C, different concentrations of HAase (0–3.6 U/mL) were assayed at various total protein concentrations. The lower detection limit was 3 U/mL. After adjusting the final protein concentration to 50 ng per reaction mixture (using BSA), the detection limit was 2.4 U/mL (Fig. 3). The λmax of AuNPs was initially located at 530 nm, and upon addition of HA the AuNPs aggregated with shift in λmax to a higher wavelength (620 nm).The absorbance at 530 nm decreased and the absorbance at 620 nm increased. The presence of HAase activity prevented AuNPs aggregation maintaining higher absorbance at 530 nm and lower absorbance at 620 nm. The colorimetric response and the wavelength change are displayed in (Fig. 4). 3.4. Qualitative detection of HAase activity in clinical urine samples using cationic AuNPs

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the results of AuNPs assay shown in (Fig. 5a). The sensitivity of zymography was 65%, while the specificity was 96.1%.

3.6. Quantitative determination of HAase activity in clinical urine samples using AuNP assay In the developed colorimetric assay, results obtained have revealed that the color change at a rate that was directly related to the amount of HAase used in the assay. The assay offers the possibility of quantitative estimation of HAase activity. To quantify HAase activity, a standard curve was drawn in which the ratios of spectral absorbance A530/A620 of the reacted AuNP solutions after 1 h reaction time were plotted as a function of the corresponding standard HAase activities normalized to protein concentration (mU/ng protein). The ratio of spectral absorbance A530/A620 of the reacted AuNP solution for each urine sample was calculated and the HAase activity of each sample was determined from the standard curve. Supplementary Fig. S4 shows the plot of the values of A530/A620 of AuNPs against the HAase activity (0–360 mU/ng

The assay has been successfully used for direct qualitative detection of HAase activity in normal urine samples spiked with the enzyme and urine samples collected from all individuals enrolled in this study. 33 malignant samples out of 40 were positive, with a sensitivity of 82.5%. On the other hand, 15 normal samples out of 15 samples were negative, while 10 benign samples out of 11 were negative and 1 sample was false positive. The specificity of the developed assay was 96.1%. 3.5. HAase activity assay by zymography Assays of HA substrate gel zymography were performed to demonstrate the activity of HAase in spiked samples and urine samples collected from all individuals enrolled in this study. The representative HA zymography (Fig. 5b) presented predominant HAase activity as clear bands at 55 kDa in case of HAase standard and malignant samples, while no bands appear for normal or benign samples. The results of zymography were matched with

Fig. 4. UV–vis absorption spectra for cationic AuNPs. Red line represents raw CTAB stabilized AuNPs (46 nm) with λmax at 530 nm. Blue line represents aggregation of cationic AuNPs on negatively charged HA with red shift and broadening of the peak. The green line and black line represent the spectral profile of cationic AuNPs þ HA in the presence of 3.6 U/mL and 2.4 U/mL HAase; respectively, with adjusted final protein concentration at 50 ng per reaction. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Fig. 3. Determination of the lower detection limit of the colorimetric assay for HAase activity using cationic AuNPs after 1 h incubation time at 37 1C. (a) Detection limit without adjusting the final protein concentration was 3 U/mL. (b) Detection limit after adjusting the final protein concentration at 50 ng per reaction mixture was 2.4 U/mL. (c) Bar chart plot for the ratios of spectral absorbance A530/A620 of reacted AuNP solutions against the corresponding HAase activities with and without adjusting the final protein concentration, red and blue bars respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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Fig. 5. (a) Colorimetric urinary HAase assay using cationic AuNPs. Each tube contained 5 mL of urine supernatant (  50 ng of protein) mixed with 15 mL of 3 mg/mL HA solution and was incubated at 37 1C for 1 h. After stopping the enzymatic reaction, 100 mL of colloidal AuNPs was added to each mixture and the color was observed within 1 min with the naked eye. Positive reaction is shown in the standard HAase (1) and malignant samples (5, 7 and 10), while negative reaction is shown in normal samples (3 and 4) and benign samples (6, 8 and 9). (b) Zymography analysis for urinary HAase. Lane 1 is the standard HAase (55 kDa). Lane 2 is the Bio-Rad prestained standard marker (7.1–209 kDa). Lanes 3 and 4 represent normal urine samples. Lanes 6, 8 and 9 represent benign urine samples. Lanes 5, 7 and 10 represent malignant urine samples.

protein) and a linear relation can be found (y¼0.006xþ 0.379, R2 ¼0.944).

4. Discussion The unique optical properties of AuNPs have allowed the development of several colorimetric biosensing systems for detection of disease biomarkers such as proteins and nucleic acids (Azzazy et al., 2006; Radwan and Azzazy, 2009). Many of the AuNP assays are still at the stage of proof-of-concept, because AuNP based systems are to a certain extent unstable in complex biological media (Agasti et al., 2010).This instability mainly due to presence of many interfering substances in biological specimens that may lead to non-specific aggregation of AuNPs. Here, we present a simple colorimetric assay for direct detection of HAase activity in clinical urine samples using cationic AuNPs. Many technical optimizations have been introduced to overcome the instability of cationic AuNPs in urine samples. The principle of this work is based on charge interaction between long chain polyanionic HA and cationic AuNPs leading to formation of gold aggregates with red to blue color shift. High concentrations of HAase give the positive results even at zero minute incubation time at 37 1C as all HA molecules are believed to be electrostatically sequestered by HAase molecules (positively charged at pH 4.3) and upon addition of AuNP solution no aggregation occurs and the solution remains red in color. This could be explained as the binding of HAase to HA involves electrostatic interaction, so large number of positively charged HAase molecules compete with cationic AuNPs for binding to HA. While at low concentrations of HAase, incubation at 37 1C is required to allow HAase molecules to digest all HA into small fragments and upon addition of AuNPs the solution remains red because smaller HA fragments are unable to aggregate the cationic AuNPs. Assay needs optimization for the concentration of both HA and AuNP solution. Results obtained have shown that at a given concentration of AuNPs, both high and very low concentrations of HA solutions did not aggregate the AuNPs. This could be explained

as, highly concentrated HA solution ( 43 mg/mL) would keep the inter-particle distance between AuNPs larger than the average particle diameter preventing the aggregation of the AuNPs. On the other hand, very low concentration of HA (o3 mg/mL) was not enough to aggregate all AuNPs in solution. Previous studies (Asteriou et al., 2006; Lenormand et al., 2007, 2008, 2009; Deschrevel et al., 2008) have reported the effect of HA–protein complex on modulating HAase activity. HA can form two types of complexes with HAase; the first is the conventional catalytic enzyme–substrate complex involving hydrogen bonds, electrostatic and Van der Waals interactions. The second is the non-specific complex that HA can form with different proteins such as BSA, lysozyme and HAase only through electrostatic binding. HA digestion by HAase is strongly suppressed at low HAase over HA concentration ratio because long chains of HA can form electrostatic and non-catalytic complexes with HAase. While, at higher concentrations of HAase, higher hydrolysis rates were obtained due to the excess free and active HAase molecules. At pH 4.0, non-catalytic proteins like BSA can compete with HAase for electrostatic binding with HA liberating HAase molecules which then recover their catalytic activity. Accordingly, the total final protein concentration was adjusted at 50 ng/reaction. Although the difference in detection limit was not significant (2.4 U/mL vs. 3 U/mL), the adjustment of final protein concentration in reaction mixture was important to ensure reproducibility of the assay. The assay has been successfully used to detect the HAase activity in normal urine samples spiked with the enzyme and urine samples obtained from all individuals enrolled in this study. The working principle of our assay for detection of HAase activity in clinical urine samples is schematically represented in Fig. 6. Generally, a big challenge with the use of cationic AuNP based biosensing systems for detection of target analytes in biological samples is the presence of wide varieties of negatively charged interfering substances (as nucleic acids and glycosaminoglycans) that may cause nonspecific aggregation giving false negative reaction. Therefore, the removal of anionic moieties that may be present in clinical samples and could interfere with the assay was essential. Many protocols were tried and the best one was the use of water insoluble polycationic polymer like chitosan. High molecular weight chitosan is insoluble in urine sample and in acidic pH acquire positive charges. So, it can successfully agglomerate all negatively charged moieties present in the sample without adsorbing positively charged HAase molecules. This effect of chitosan is consistent with previously reported use of chitosan as a sequestering agent for anionic moieties in wastewater treatment (Sridhari and Dutta, 2000). All samples were tested with AuNPs after chitosan treatment to ensure the removal of all interfering substances before performing the assay (Supplementary Fig. S3). Comparing the assay results to conventional HA substrate gel zymography for qualitative detection of HAase activity, the sensitivity of the developed assay was 82.5%, while the sensitivity of zymography was 65%. The specificity of both assays was 96.1%. The developed AuNPs assay was more sensitive than the traditional zymography method (82.5% vs. 65%). The developed assay has a shorter turnaround time (2 h) compared to zymography (48 h). Quantitative estimation of HAase activity using the developed AuNP assay in malignant, benign and normal groups was performed. Receiver operating characteristics (ROC) curve was created by Statistical Package for the Social Sciences software (SPSS version 16) to determine the threshold value for optimal sensitivity and specificity. Accordingly, the best cut off value by considering the benign and healthy normal groups non-malignant control group for HAase activity was 93.5 mU/ng protein, at which the sensitivity was 90% and the specificity was 80.8% and the area under the curve was 0.950 (Supplementary Fig. S5). The quantified urinary HAase activity was significantly higher in the malignant

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very short time compared to zymography (48 h). Being able to discriminate between malignant and non-malignant samples rapidly and without any costly or complex instrumentation, the new HAase AuNP assay is proposed as a valuable tool to aid bladder cancer diagnosis.

Acknowledgments Authors thank Mr. Sherif Shawky and Dr. Marwa Matboly for their technical assistance. This study was funded by the Egyptian Academy of Research and Technology, the Science and Technology Center, Project 21/2 to Prof Sanaa Eissa and grants from Yousef Jameel Science & Technology Research Center at AUC to Prof. Hassan Azzazy.

Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.bios.2013.10.024. References

Fig. 6. Schematic illustration of cationic AuNPs based colorimetric assay for direct detection of HAase activity in urine samples. First, urine samples buffered at pH 4.3 are treated with chitosan and tested with cationic AuNPs to ensure the removal of interfering moieties. Then, 5 mL of urine supernatant (  50 ng of protein) is mixed with 15 mL of 3 mg/mL HA solution and incubated at 37 1C for 1 h. After stopping the enzymatic reaction, 100 mL of colloidal AuNPs is added to each mixture and the color is observed within 1 min with the naked eye. Positive urine sample gives red color (no aggregation), while negative one gives blue (aggregation).

group (mean¼147.95 749.61) as compared with benign (mean¼ 82.63 723.74) and normal control groups (mean ¼ 74.93 710.74), p r0.01 (Table S1).

5. Conclusions In the present study, a colorimetric AuNP assay for rapid and sensitive detection of urinary HAase activity has been developed. The unique optical properties of AuNPs allowed the differentiation between malignant samples with positive HAase activity and nonmalignant samples through visualization of change in solution color from red (positive sample) to blue (negative sample). Pretreatment of samples with the polycationic chitosan, which was able to agglomerate all negatively charged interfering moieties from the samples prior to performing the assay, allowed the use of this new assay successfully in detecting HAase activity in clinical samples. Additionally, UV–vis spectroscopy was used to quantitatively estimate the HAase activity. HAase activity could be detected with higher sensitivity in malignant samples within 2 h which is a

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Direct detection of hyaluronidase in urine using cationic gold nanoparticles: a potential diagnostic test for bladder cancer.

Hyaluronidase (HAase) was reported as a urinary marker of bladder cancer. In this study, a simple colorimetric gold nanoparticle (AuNP) assay was deve...
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