water research 43 (2009) 1425–1431

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Detection of pathogen based on the catalytic growth of gold nanocrystals Xin Xing Li, Cuong Cao, Se Jong Han, Sang Jun Sim* Nano-optics and Biomolecular Engineering National Laboratory, Department of Chemical Engineering, Sungkyunkwan University, 300 Chunchun-dong, Jangan-gu, Suwon 440-746, Republic of Korea

article info

abstract

Article history:

A homogenous detection of pathogen (Giardia lamblia cysts) based on the catalytic growth

Received 19 September 2008

of gold nanoparticles (AuNPs) has been studied. In this study, centrifugal filters were

Received in revised form

employed as tools to concentrate and separate the pathogen cells, and moreover amplify

11 December 2008

the detection signal. The catalytic growth of gold nanoparticles was verified to be positively

Accepted 12 December 2008

related to gold seeds concentration. On this basis, homogenous detection of the pathogenic

Published online 25 December 2008

bacteria in liquid phase was established by means of conjugating antibody to gold seeds.

Keywords:

as low as 1.088  103 cells ml1. The additional nonspecific binding tests were also con-

Gold nanoparticles

ducted to verify the detection specificity. This sensing platform has been proved to be

Catalytic growth

a sensitive, reliable and simple method for large-scale pathogen detection, and provide

Giardia lamblia cysts

valuable insight for the development of gold nanocrystals based colorimetric biosensors.

Under the given experimental condition, detection limit of G. lamblia cysts was determined

ª 2008 Elsevier Ltd. All rights reserved.

1.

Introduction

The intestinal protozoan Giardia lamblia involved in infectious gastroenteritis is a potential worldwide threat to human being’s health. Outbreaks of giardiasis occurring via fecal–oral transmission are associated with consumption of contaminated food and drinking water. In order to develop a simple and sensitive technique to detect G. lamblia, a diversity of methods have been intensively investigated such as microscopical diagnosis (Mank et al., 1997), enzyme immunoassays (Garcia and Shimizu, 2000), direct fluorescent-antibody staining test (Garcia et al., 1992), as well as real-time PCR (Verweij et al., 2003). However, all of these methods are expensive, time costing and labor-consuming. Highly sensitive, fast and reliable method for detection of G. lamblia still needs unremitting effort in the researches of environmental and health-care. Currently, application of nanometer sized materials in the detection of biointeraction events has attracted great interests

(Willner et al., 2007; Daniel and Astruc, 2004; Wang, 2005). AuNPs have been applied in various fields such as biochips and biosensors based on their stability, low cost, facile preparation and modification (Jana and Ying, 2008; Jana et al., 2007). The favorable biocompatibility and high surface to volume ratio enable AuNPs as an ideal material for adsorption of biomolecules without compromising their biological activities. Antigens or antibodies functionalized AuNPs can serve as optical labels (Ambrosi et al., 2007), electrochemical markers (Zhou et al., 2006), surface plasmonic amplifiers (Cao and Sim, 2007; Kang et al., 2008) or signal transfer mediator (Nam et al., 2003) for the quantitative analysis of ligands. AuNPs in combination with other signal generators could doubly amplify the signal (Li et al., 2008). Utilizing the property of AuNPs for catalytic growth to amplify the immunoassay signal is an emerging field attracting extensive attention. The best well-known example is silver enlargement, also called as silver staining, has been well developed and widely applied in the biological detection (Jana

* Corresponding author. Tel.: þ82 31 290 7341; fax: þ82 31 290 7272. E-mail address: [email protected] (S.J. Sim). 0043-1354/$ – see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2008.12.024

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et al., 2007; Xu et al., 2007; Chen et al., 2007). Silver staining utilizes AuNPs as catalytic probes to transfer biorecognition into optical signal. However, although the silver staining method allows us to directly observe the immunoassay signals on a glass chip or a nitrocellulose strip by naked eyes or specific analytical equipment, the sensitivity is much lower than those enhanced by fluorescent, radioactive, and colorimetric assays. Recently, instead of silver staining, gold salt was utilized to enlarge immune AuNPs as an alternative strategy for the catalytic growth based detection. Ma and Sui (2002) have developed a platform using a mixture of HAuCl4 and NH2OH$HCl to enlarge AuNPs immobilized on the nitrocellulose strip to detect human immunoglobulin G (h-IgG). The detection limit approximately achieved 10 pg ml1 and rivaled conventional fluorescent, radioactive, and enzyme-colorimetric methods. On this basis, Su developed a novel detection platform by immobilizing gold immunoprobes on glass slide and the detection limit was lowered down to 0.1 pg ml1 (Su, 2006). Due to the attractive potential, this research has been applied in the detection of DNA (Wang et al., 2007; Yang et al., 2007), proteins (Ma and Sui, 2002; Su, 2005, 2006) and cells (Ma et al., 2002). However, most of the studies were carried out in solid phase. The detection of protein biomarker based on homogenous growth of Au nanocrystals in solution phase has been introduced by our group (Cao et al., in press). In the solution phase, the immuno-recognition event is translated into the gold nanoparticles growth signal which can be intuitively recognized by an unaided eye, or quantitatively measured by an UV/vis spectrophotometric analysis. This assay was highly sensitive, robust, simple, and it has great potential to detect other biological interactions. Therefore, it is a significant attempt to utilize those advantages to realize a homogenous detection of pathogen cells such as G. lamblia cysts. In this study, the whole experiment took advantage of centrifuge filters for cell separation and collection. During the immunoassay, target pathogen cells were captured by antibody functionalized AuNPs, and then collected and separated by centrifuge filters. Those AuNPs remained on the cells were proportional to analytic pathogen cells and furthermore played a role of color developer. The quantitative detectable color development was derived from the catalytic growth of AuNPs. All the immunoassays were completed in a homogenous liquid phase, and a detection limit of 1.088  103 cells ml1 was achieved. Complementary nonspecific immunoreaction also supported the specificity of this detection system. The results demonstrated that the catalytic enlargement of AuNPs was a fast, sensitive and reliable method that might be a promising method for analogous detection of other pathogens.

2.

Materials and methods

2.1.

Materials and instrumentation

Mouse anti-G. lamblia IgG was purchased from AbD Serotec Inc., USA. Oocysts of Cryptosporidium parvum and cysts of G. lamblia were provided by Waterborne Inc., USA. Chlamydomonas reinhardtii and Chlorella zofingiensis were kindly provided

by the University of Texas at Austin and KRIBB (Korea Research Institute of Bioscience and Biotechnology), respectively. Hydrogen tetrachloroaurate (III) trihydrate (HAuCl4$3H2O, 99.9%), cetyltrimethylammonium bromide (CTAB), ascorbic acid (AA), sodium citrate, 0.01 M phosphate buffer saline pH 7.4 with Tween 20 (PBS buffer), bovine serum albumin (BSA) and ultrafree-MC microcentrifuge filters with pore size of 0.45 mm were from Sigma–Aldrich. Other essential inorganic reagents were supplied by Pierce, Sigma, Aldrich, or Fluka unless special stated. All chemicals were used as received, and all of the chemical solutions were prepared in ultra pure water (18.2 mU) when needed. The life science UV/vis scanning spectrophotometer (DU 730) used in this study was purchased from Beckman Coulter, Inc. Nanostructure characterization was performed by highresolution transmission electron microscopy (HRTEM, model JEOL JEM-3010). The multipurpose refrigerated centrifuge (VS5500CFN, 15CFN) was from Vision Scientific Co., Ltd.

2.2.

Synthesis and characterization of AuNPs

AuNPs were prepared using sodium citrate as the reductant and stabilizer according to the literature (Ambrosi et al., 2007) with slight modification. Briefly, 0.5 ml of 50 mM HAuCl4 solution was mixed with 100 ml fresh water. The mixture was stirred vigorously and heated until boiling; 2.5 ml of 1% sodium citrate solution was added into the mixture. A continuous change in color from colorless to red was observed. After the color change ceased, kept stirring for another 30 min, removed from the heat and then stirred for another 15 min. When the solution was cooled down to room temperature, ultra pure water was added to compensate the evaporated part. The quality of colloidal gold could be simply evaluated by observing the solution color with naked eyes. UV/vis spectrophotometer and HRTEM were also utilized for morphology characterization of AuNPs.

2.3.

Preparation of Au immunoprobes

The Au immunoprobes were prepared mainly by utilizing the hydrophobic adsorption and electrostatic interaction between IgG macromolecules and AuNPs. Before preparation, the minimal amount of protein to stabilize AuNPs should be determined. Colloidal gold solution was firstly adjusted to pH 9.0 with 100 mM Na2CO3 solution, 500 ml of colloidal AuNPs was subsequently mixed with 50 ml of mouse anti-G. lamblia monoclonal IgG antibody solutions in various concentrations up to 0.1 mg ml1, and then incubated at room temperature for 20 min. Finally, 300 ml of 10% NaCl was added and rapidly mixed. Unsuccessful adsorption of IgG to AuNPs was indicated by the color change from red to light blue due to aggregation of the AuNPs. Thereupon, the minimum amount of the antibody could be easy to identify through color observation since such an amount is enough to prevent the AuNPs from aggregation. The overall process to prepare immunoprobes was referred to the literature (Ambrosi et al., 2007) with some modifications. A solution containing minimal amount of necessary IgG plus 10% was added into 10 ml of pH-adjusted AuNPs and stirred for 15 min. Afterwards, BSA was added to final

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concentration of 1 mg ml1 for blocking the AuNP surface to prevent unspecific adsorption of other proteins. After the incubation, the antibody labeled AuNPs were collected and rinsed with PBS buffer by centrifugation at 10,000 rpm for 40 min at 4  C. After two cycles of rinsing, the Au immunoprobes were resuspended in 10 ml PBS buffer. The gold immunoprobe solution must be filtered through 0.45 mm filters before storage at 4  C.

2.4.

Preparation of growth solution for Au enlargement

The growth solution was prepared according to our previous study (Cao et al., in press). Briefly, 0.05 ml aqueous solution of 0.05 M HAuCl4 was mixed with 10 ml 0.1 M cetyltrimethylammonium bromide solution, and heated until the solution turned a clear orange color. After cooling down to room temperature, as soon as 1 ml 0.1 M ascorbic acid solution was added, solution color immediately disappeared. Now the growth solution was ready for use.

2.5.

Immunoassays

The whole immunoassay was completed with the assistance of centrifuge filters. Before carrying out the experiment, all the centrifuge filters were blocked with 1% BSA to avoid any potential unspecific adsorption of gold probes, and then rinsed for 3 times with PBS buffer. Fig. 1 shows schematic description of this process. Briefly, 10 ml of solution

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containing G. lamblia cysts in various concentrations was concentrated by centrifuge filters and the residue was resuspended in 150 ml gold immunoprobe solution. The immunoreaction between gold immunoprobes and target cells was accomplished after 1 h incubation at 37  C C. Then, the free gold immunoprobes were removed by centrifugation, and the residue was rinsed with PBS buffer for 3 times. Finally, the residue was resuspended in 100 ml ultra pure water and transferred into 900 ml freshly prepared growth solution. The color development driven by gold catalytic growth was recorded by UV/vis spectrophotometer.

3.

Results and discussion

3.1.

Characterization of AuNPs

Preparation of AuNPs is crucial to the stability of the gold immunoprobes. Owing to the fact that the stability of immunoprobes strongly depends on size and shape of the AuNPs, synthesis of monodispersed round AuNPs is the precondition of the whole immunoassay. The quality of gold immunoprobes could be evaluated by UV/vis spectrophotometric analysis due to the surface plasmon excitation of the AuNPs. The synthesized AuNPs have typical maximum absorbance at 519 nm, and the maximum peak was slightly shifted to 527 nm after the physical adsorption of IgG antibody to the gold surface (data not shown). HRTEM was utilized for more

Fig. 1 – Schematic illustration for the whole experimental strategy: (1) sample transfer; (2) sample concentration; (3) immunoassay incubation; (4) free gold probes separation; (5) sample transfer; (6) gold catalytic growth; and (7) UV/vis spectrophotometer characterization.

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Fig. 2 – The high-resolution TEM image (a) before and (b) after catalytic growth. The scale bar is 20 nm and 50 nm, respectively.

direct characterization of the synthesized AuNPs. As showed in Fig. 2(a), the spherical and monodispersed colloidal AuNPs were obtained with a size of about 15  1 nm, their smooth surface is supposed to facilitate the adsorption of protein.

3.2.

Growth of AuNPs in aqueous solution

The catalytic enlargement of AuNPs has been widely used for monitoring small organic molecules, proteins, cells and other biological analyte (Ma and Sui, 2002; Su, 2005, 2006; Wang et al., 2007; Yang et al., 2007; Ma et al., 2002; Zayats et al., 2005). In the enlargement process, reductant provides electrons to reduce Au3þ complex to Auþ, which could be subsequently reduced into Au0 at the surface of small Au seeds in the presence of metal gold (Sau and Murphy, 2004). This results in the enlargement of gold seeds accompanied by increased optical absorbance. Herein, the AuNPs act as nuclei and catalysts for further growth of the Au nanocrystals. Up to now, NH2OH (Ma and Sui, 2002; Su, 2005, 2006), H2O2 (Zhou et al., 2006; Shang et al., 2008), NADH (Wang et al., 2007; Yang et al., 2007; Xiao et al., 2005, 2004) and even biomacromolecules (Willner et al., 2006) have been used as reductant during the gold enlargement process. As conventional reductant, AA has been applied in the synthesis of different size of AuNPs (Jana et al., 2001). In the presence of stabilizer CTAB and the Au nuclei, the whole catalytic enlargement process can be simply demonstrated by an equation below (Cao et al., in press):

low concentration, it was seemingly that there is no help to lower down the detectable limit even prolong the detection time. In the view of fast principle in design, 20 min was chosen as time parameter. In another aspect, the signal increased dramatically with elapsed time, which was the reason why the catalytic enlargement can be utilized as amplifier during biomaterial detection. TEM characterization also revealed the fact of gold enlargement and provided us a visualized image of morphology change during enlargement process. Fig. 2 shows the typical images of AuNPs (a) before and (b) after growth. As shown in Fig. 2, the as-prepared AuNPs (a) were of uniform spherical shape and the size was around 15  1 nm in diameter. After treatment of growth solution (b), the diameters of these AuNPs were enlarged to nearly 70 nm. One thing should be mentioned was that the shape of the AuNPs after growth was not as homogenous as shown in Fig. 2(b), some rod and irregular shaped nanoparticles were generated after catalytic enlargement. Owing to the stabilization benefit of CTAB, the enlarged AuNPs were able to be well-dispersed in solution.

AA

Au3þ þ Au0n¼1 ðseed; atom ¼ 1Þ þ 3e ! Au0n¼2 The growth process is positively related with seed concentration (Su, 2005). The higher concentration of gold seeds leads to faster growth speed. During the growth process shown in Fig. 3, the plasmonic peak had a gradual red-shift and became broader as the time goes. We chose 550 nm of the wavelength as an analytical parameter for quantitative justification of the nanoparticles growth throughout the assay since the maximum absorbance peaks were stably centered about 550 nm after 20 min of the catalytic growth process. At the same time, due to the unidentified difference in the range of

Fig. 3 – UV/vis spectrophotometer scanning during the catalytic growth process.

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3.3. Detection of G. lamblia cysts using catalytic growth of AuNPs in liquid phase By analyzing the parameters about the gold catalytic growth, the assay was extended to a practical detection of pathogen. In this study, AuNPs worked as signal generators and amplifier while the centrifuge filters were utilized as requisite tool to separate nanoparticles from huge sized pathogens. The size of G. lamblia cysts is 6–10 mm while our filter pore size is 0.45 mm, meanwhile, the conjugation of AuNPs and IgG gives a size around 25 nm in diameter (Ambrosi et al., 2007), therefore it is possible to fully utilize filters to separate and concentrate cells. In common sense, our system can collect cells in larger volume and give higher sensitivity, however, considering real experimental condition, 10 ml sample was chosen as work volume. As shown in Fig. 1, the centrifuge filters were mainly used in steps (2) and (4). Analyte cells were concentrated and resuspended in 150 ml gold immunoprobe solution. After incubation and removing free gold immunoprobes in step (4), the residue was resuspended in 100 ml water and transferred into 900 ml growth solution. At this stage, gold seeds began to act as nuclei and catalyzed the deposition of metal atoms. Samples in different concentrations gave different growth

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speed and finally rendered signals in different intensity at analytical time point. The quantitative assessment of the analytical concentration was based on the absorbance measurement at 550 nm by UV/vis spectrophotometer. The 20th min was chosen as analyzing time parameter. A broad concentration range from 0 to 105 cells ml1 was selected for the primary investigation for pathogen. Fig. 4(a) shows calibration curves generated by plotting absorbance at 550 nm wavelength against analytic pathogen cell concentration. The error bars represent the standard deviation of concentrations ranging from 0 to 10 cells ml1. The plots indicated that the signals were significantly varying among different concentration of G. lamblia. By further detailed investigation, a linear relationship was observed in the range from 103 to 104 cells ml1. The linear range obtained then allowed for quantitative detection. Fig. 4(b) provides the linear range of the immune response to analytic concentration. A linear regression equation was calculated as y ¼ 105 x þ 0.0159 (R2 ¼ 0.0989), where x and y stand for analytic concentration and UV/vis spectra absorbance signal, respectively. The standard deviation of zero concentration was 3.625  103, therefore, the LOD (limit of detection) was fixed as 1.088  103 cells ml1. Though this detectable limit was not as low as literature (Ma et al., 2002), but as one quantitative detection method in homogenous phase, its simplicity and applicability gave considerable contributions in pathogen diagnostics. The image of gold immunoprobes binding to G. lamblia cysts will be the authentic proof of immune event. Therefore, HRTEM provided figures of this event. In Fig. 5, it was clear that the elliptic silhouette (the dark area) of the cell wall (corresponding part of cysts was marked) was intensively covered due to the interaction between the antigen located on the cell wall surface and gold immunoprobes. The scale bars were (a) 500 nm and (b) 100 nm, respectively. At the same time, there was almost no AuNPs could be seen beyond the cell region in sight, which means that the free AuNPs had been removed efficiently. Therefore, high background noise or even the false positive signal resulted from the growth of excessive gold probes was prevented in the presence of AA and CTAB. In Fig. 4(b), low signal of 0 cells ml1 compared to other concentrations also demonstrates the effectivity of separation.

3.4. Investigation of nonspecific binding of different pathogens in the detection assay

Fig. 4 – Calibration curve corresponding to the absorbance at l [ 550 nm against to analytical cell concentration. (a) The wide range detection from 0 to 105 cells mlL1 and (b) the linear relationship range from 0 to 104 cells mlL1.

It is known that a huge amount of various microorganisms live in the natural water source and whether these microorganisms render cross-linking to gold immunoprobes during detection process and lead to aberrance of the final results should arose our special attention. Besides, whether the large size of these microorganisms could block filter pores is another suspicious point. Considering these unclear factors, it is necessary to ensure the specificity of our detection system. Three kinds of microorganisms (C. parvum, C. reinhardtii and C. zofingiensis) existing in water were chosen as references for this investigation. All the microorganisms were present in the concentration range from 103 to 105 cells ml1. Under exactly identical experimental condition, at least three replicates were carried out. In Fig. 6, it was obvious to distinguish Giardia

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Fig. 5 – Images of high-resolution TEM for the visualization of gold probes sticking to the cell wall surface of Giardia lamblia cysts. The scale bar is (a) 500 nm and (b) 100 nm, respectively. The inset shows the morphology of G. lamblia cysts.

method and offers cherished experience for future development of biosensors.

Acknowledgements This work was supported by the Korea Science and Engineering Foundation (KOSEF) National Research Laboratory (NRL) Program grant funded by the Korea government (MEST) (grant no. R0A-2008-000-20078-0) and Core Environmental Technology Development Project for Next Generation funded by the Ministry of Environment of the Republic of Korea.

references Fig. 6 – Nonspecific immunoreaction test of different microorganisms for the detection assays.

from the other kinds of microorganisms since these microorganisms just gave fluctuated signal in low level as increased concentration. That means there was almost no cross-interaction between gold probes and other large size microorganisms in water. As a whole, it strongly supported that our system had high specificity to G. lamblia cysts.

4.

Conclusions

A novel platform was described for homogenous detection of pathogen (G. lamblia cysts) using AuNPs catalytic growth assisted by centrifuge filters. Multiple signal enhancement in the experiment facilitated achievement of a final detectable limit of 1.088  103 cells ml1 for pathogen. The linear relationship between signal intensity and analytic cell concentration was also well investigated in the concentration range of 103–104 cells ml1. Nonspecific test showed the high specificity of this method and ensured its availability for practical application. It was proved that this was a rapid and valuable

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