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Biosensors and Bioelectronics 25 (2010) 2534–2538

Contents lists available at ScienceDirect

Biosensors and Bioelectronics

journa l homepage: www.e lsev ier .com/ locate /b ios

hort communication

competitive immunochromatographic assay based on a novel probe for theetection of mercury (II) ions in water samples

u Zhou ∗, Yuanyuan Zhang, Fengguang Pan, Yansong Li, Shiying Lu, Honglin Ren,ingfeng Shen, Zhaohui Li, Junhui Zhang, Qijun Chen, Zengshan Liu ∗

ey Laboratory for Zoonosis Research, Ministry of Education, Institute of Zoonosis, Xi-an Road 5333, Jilin University, Changchun, Jilin 130062, PR China

r t i c l e i n f o

rticle history:eceived 19 February 2010eceived in revised form 28 March 2010ccepted 2 April 2010vailable online 13 April 2010

a b s t r a c t

Mercury ions (Hg2+) are one of the most dangerous pollutants. Even at low concentration, it causes seri-ous environmental and health problems. Current methods for the detection of Hg2+ in environmentalsamples are tedious and time consuming because they require sophisticated instrumentation and com-plicated sample pre-treatment processes. In this work, a novel probe with high selectivity towards Hg2+

was synthesized and a one step competitive immunochromatographic assay based on the probe for the2+

eywords:ercury (II) ions

robeisual detectionest strip

detection of Hg was developed and applied for water samples. The detection conjugate was immobi-lized on one end of the nitrocellulose membrane (detection line) and anti-BSA polyclonal antibody wasimmobilized on the other end of the membrane (control line). Hg2+ in samples competed with the probeto bind with immobilized detection conjugate. The visual detection limit of Hg2+ in spiked water sampleswas found to be about 1 ppb. The qualitative assay can be performed within 15 min. The advantages ofthe technique are rapidity, low cost and without the need of any equipment and complicated samplepreparation.

. Introduction

Mercury ions (Hg2+) are considered a highly toxic element andts contamination is a global problem. Exposure of mercury event low concentration results in health damage to their inhabitants.t passes biological membranes very easily, binds to enzymes andisrupts vital functions (Marx and Hock, 2000). The World Healthrganization (WHO) recommended water quality guidelines of less

han 6 ppb (�g/L) for inorganic mercury (Takahashi et al., 2009). Thenited States Environmental Protection Agency (EPA) acceptable

imit for Hg2+ is 2 ppb in drinking water (Díez-Gil et al., 2008).Therefore, the detection of Hg2+ level in water samples is clearly

mportant. To date, many current techniques for mercury screen-ngs, such as enzymatic detection (Prakash et al., 2008), liquidhromatographic analysis (Dago et al., 2009), fluorescence detec-ion (Wang et al., 2009), colorimetric detection (Takahashi etl., 2009), Nile blue-based chemodosimeter detection (Lee et al.,

009a), electrical detection with AlGaN/GaN high electron mobil-

ty transistors (Chen et al., 2008a), inductively coupled plasmaass spectrometry (ICP-MS) (Minnich et al., 2008), require expen-

ive and sophisticated instrumentation, specific technical skills

∗ Corresponding author. Tel.: +86 0431 87836718/1363 4318992.E-mail addresses: zhouyu69@sina.com (Y. Zhou), zsliu1959@sohu.com (Z. Liu).

956-5663/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.bios.2010.04.003

© 2010 Elsevier B.V. All rights reserved.

for complicated sample pre-treatment processes and machineoperation and/or has cross-sensitivity towards other metal ions.Consequently, simple, cheap, efficient, highly sensitive and selec-tive on-site analytical methods have been demanded for quickjudgment of water.

It is one of the key steps to synthesize detection probe for thedevelopment of an on-site screening method for Hg2+. In resentyears, some available detection probes such as fluorescence probes(Lee et al., 2009b; Cai et al., 2006; Chen et al., 2008b), optical fibreprobe (Pérez-Hernández et al., 2009) and chemical probe (Pérez-Hernández et al., 2009) for detection of Hg2+ have been synthesized.However, the detection methods based on these probes requireexpensive equipment and skilled analysts. The test strip platformbased on gold nanoparticle probe is well suited for on-site screen-ing, which has been used for some environmental contaminants (Liet al., 2009; Zhou et al., 2009a, 2010; Gas et al., 2010) and pesti-cides (Gui et al., 2008; Shi et al., 2008; Blazková et al., 2009). Toour best knowledge, any use of test strip platform based on goldnanoparticle probe for Hg2+ detection has not been published upto now.

In this paper, a novel detection receptor with high selectiv-ity towards Hg2+ was synthesized and a gold nanoparticle-baseddetection probe was combined. A test strip platform based on thedetection probe was developed for Hg2+ detection in water sam-ples.

Y. Zhou et al. / Biosensors and Bioelectronics 25 (2010) 2534–2538 2535

F trip. (p are de

2

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ig. 1. Synthesis procedure of the conjugates and schematic diagram of the test srobe conjugate, (C) schematic diagram of the test strip. Details on the preparation

. Materials and methods

.1. Materials and chemicals

10-Undecenoic acid, mercuric acetate, methanol, carbon disul-de, iso-butyraldehyde, NaBH4, CS2, BSA, N,N-dimethylformamideDMF), N-hydroxysulfosuccinimide (NHS) and carbodiimide (EDC)ere purchased from Beijing Chemical Reagent Co. Ltd. (Beijing,hina). Bovine serum albumin (BSA) chloroauric acid (HAuCl4) andrisodium citrate were obtained from Shanghai Chemical ReagentsShanghai, China). High-flow nitrocellulose membrane, glass fibre,nd absorption pad were purchased from Millipore (Bedford, MA).

ll other chemicals were of analytical grade or better and werebtained from Beijing Chemical Reagent Co. (Beijing, China). Col-oidal gold nanoparticles (20 nm mean diameter) were preparedccording to the method by the researchers themselves (Zhou etl., 2009a).

A) Synthesis procedure of the detection conjugate, (B) synthesis procedure of thescribed in the text.

2.2. Preparation of anti-BSA polyclonal antibody

Two New Zealand rabbits weighing about 2.5 kg were used forraising polyclonal antibodies according to the procedures in litera-ture (Watanabe et al., 2010). Briefly, 1 mg of BSA diluted in 0.5 mLof PBS was thoroughly emulsified with an equal volume of com-plete Freund

′s adjuvant. The emulsion in the initial immunization

was intradermally injected in footpad sites. The booster immuniza-tion was made intradermally and subcutaneously at multiple siteson the back of the rabbit with 1 mg of BSA (0.5 mL) and the samevolume of Freund

′s incomplete adjuvant at 2-week intervals after

the initial immunization. On the 7th day after each booster, blood

sample was taken from a marginal ear vein to check the titer ofthe antibody using ELISA in 96-well polystyrene microtiter plates(Stripwell Plate 2592, Costar, Changchun, China). Well absorben-cies were read with a MK3 microplate reader (Thermo, Shanghai,China). The blood samples were coagulated for about 2 h at room

2 Bioele

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2a

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would be bound firstly with Hg , leaving fewer detection probefor detection receptor. Consequently, less detection probe wouldbe remained at the detection receptor location on the NC mem-brane. Thus, the density of the test line would be lighter than that ofthe control line (Fig. S3, strip 2–5 in supporting information). While

Table 1Cross-reactivity of the test strip with other metal ions.

Metal ions Concentrations of the metal ions (ppb)

0.1 10 50 250 1000 5000

Mn2+ +a + + + + +Ca2+ + + + + + +Co2+ + + + + + +Al3+ + + + + + +Pb2+ + + + + + +Cr6+ + + + + + +Cr+ + + + + + +Fe2+ + + + + + +Cd2+ + + + + + +Fe3+ + + + + + +Mg2+ + + + + + +Cu2+ + + + + + ±b

536 Y. Zhou et al. / Biosensors and

emperature. After being stored overnight in a refrigerator at 4 ◦C,he samples were centrifuged and the supernatants were stored inonveniently sized at −80 ◦C.

.3. Preparation of detection receptor

The detection receptor was prepared according to a modifiedethod of Szurdoki et al. (1995). A schematic description of the

reparation for the detection conjugates is shown in Fig. 1, A.riefly, solution A: 50 mg of BSA was diluted in 10 mL of PB solu-ion (0.1 M sodium phosphate-buffered saline, pH 7.2), then 20 �Lf iso-butyraldehyde was added into the solution and the mixtureas gently stirred for 4 h at room temperature. Solution B: 30 mg

f NaBH4 was diluted in 1 mL of 0.1 M NaOH solution. Solution C:alf volume of the solution B was added into the solution A andently stirred for 1 h, then another half volume of the solution Bas added and the mixture was gently stirred for 2 h. Solution D:

he solution C was dialyzed against 1 L of 0.1 M PB (pH 7.2) at 4 ◦Cor 72 h with four changes of PB to remove residuals. The pH of theolution D was adjusted to 11 with 1 M NaOH. With gently stirring,0 �L CS2 was added into pH adjusted solution D. The mixture wasently mixed for 5 h and then dialyzed against 1 L of 0.1 M PB (pH.2) at 4 ◦C for 72 h with four changes of PB to remove residuals.

.4. Synthesis of the detection probe

The schematic description of the synthesis of the probe is shownn Fig. 1B. Briefly, conjugate A: 100 mg of mercuric acetate wasiluted in 2.0 mL of methanol, then 65 �L of 10-undecenoic acidas added into the solution and incubated for 4 h at room tem-erature. And then 4 mL of DMF, 26 mg of NHS and 30 mg of EDCere added in that order. The reaction mixture was gently stirred

or 5 h at room temperature and then dialyzed against 1 L of 0.1 MB (pH 7.2) at 4 ◦C for 72 h with four changes of PB. Conjugate B:he pH of colloidal gold solution for BSA conjugation was adjustedo 8.5 with 0.1 M K2CO3. With gently stirring, 5 mL of 10% BSA solu-ion was added drop by drop to 50 mL pH adjusted colloidal goldolution. The mixture was gently mixed for 30 min and centrifugedt 20,000 × g for 30 min. After centrifugation, the gold pellets wereuspended in 5 mL dilution buffer [20 mM Tris/HCl buffer (pH 8.2)ontaining 1% (w/v) BSA] and adjusted the optical density to 5.0 at20 nm with dilution buffer and then stored at 4 ◦C for use. Con-

ugate C (detection probe): 1 mL of conjugate B was added intomL of conjugate A. The mixture was gently stirred for 4 h at room

emperature and then centrifuged at 20,000 × g for 30 min. Afterentrifugation, the pellets were suspended in 5 mL dilution buffer.he synthesized compounds were analyzed using a modificationf the methods of Zhou et al. (2009b) and the detection probe washecked with a transmission electron microscope (TEM, H-7650)Figs. S1 and S2 in supporting information).

.5. Development of the competitive immunochromatographicssay

The acyclic dithiocarbamate having presumably higher avidityor the Hg2+ (Szurdoki et al., 1995) was employed as immobi-ized ligand. The assay is based on the competition between Hg2+

in samples) and the conjugated detection probe (an organomer-ury conjugate in binding to a gold-labeled chelating conjugate).he detection probe could react with Hg2+ in sample firstly, thenemaining probe would bind with detection receptor that located

t the test line on the membrane.

The test strip was composed of a plastic packing, a nitrocelluloseembrane and three pads (sample, probe and absorbent pads). The

ads and the nitrocellulose membrane were pasted onto the adhe-ive plastic backing. The scheme of the development of the test strip

ctronics 25 (2010) 2534–2538

was shown in Fig. 1C. The membrane containing 1.5 �g of detectionconjugates (test line) and 1.8 �g of anti-BSA polyclonal antibody(control line) (in 0.1 M PB solution, pH 7.2) was cut into sections(1.5 cm × 0.5 cm) and pasted at the center of the backing plate. Thedistance between the test line and control line was about 5 mm. Theprobe pad containing the gold-labeled BSA-undecenoic acid-Ssuwas pasted by over-crossing 2 mm with nitrocellulose membraneand the sample pad was pasted by over-crossing 4 mm with theprobe pad. The absorbent pad was pasted on the other side of theplate.

2.6. Cross-reactivity

To evaluate the specificity of the probe, some metal ions suchas Ca2+, Co2+, Al3+, Ag+, Cr+, Cr6+, Fe2+, Cd2+, Fe3+, Mg2+, Mn2+, Pb2+

and Cu2+ were tested for cross-reactivity at six final concentrationsof 0.1, 10, 50, 250, 1000 and 5000 ppb for each of metal ions.

2.7. Analysis of water samples

Samples of double distilled water, a commercial mineral water,tap water, and surface water of Nanhu lake (Changchun, Jilinprovince, China) were spiked with Hg2+ at five final concentrationsof 0.1, 1.0, 10, 100 and 1000 ppb for each of samples. Particularly,tap water and lake water samples were filtered through a 0.45 �mnylon filter.

3. Results and discussion

3.1. Result judgment

While there was no Hg2+ in sample solution, the detection probewould be captured by detection receptor (test line) and anti-BSApolyclonal antibody (control line) coated on membrane. Two lineswould be appeared due to the accumulation of detection probe.The colour density of the control line and test line could be thesame and the detection probe just be used up by adjust the con-centration of the detection receptor, anti-BSA polyclonal antibodyand detection probe (Fig. S3, strip 6 in supporting information). Oncontrast, for sample solution containing Hg2+, the detection probe

2+

Ag+ + + + + ± ±a Negative result. The colour density of the test line is the same as that of the

control line.b Positive result. The colour density of the test line is lighter than that of the control

line.

Y. Zhou et al. / Biosensors and Bioelectronics 25 (2010) 2534–2538 2537

Table 2The detection of Hg2+ in different spiked water samples.

Spiked concentrations (ppb) Water samples

Mineral water Tap water Lake water PB solution

0.1 +a + + +1.0 ±b ± ± ±

10 ± ± ± ±100 ± ± ± ±

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1000 −a Negative result. The colour density of the test line is the same as that of the conb Positive result. The colour density of the test line is lighter than that of the contc Positive result. No test line is observed.

he concentration of Hg2+ is higher enough, the binding sites on theetection probe molecules would be used up. Thus the probe couldot be captured by the detection receptor. Consequently, the test

ine would disappear (Fig. S1, strip 1 in supporting information).o the positive result is judged by the appearance of two lines, andhe colour density of test line is weaker than that of the controline, or the appearance of only control line. The negative result isudged by the appearance of two lines, and the colour densities ofwo lines are the same. If there was no line or only one test lineresents in detection zone, the test could be considered as invalid.he test result judged visually could be read after 15 min. The colourensity of the test line correlated with the concentration of Hg2+

n sample in the range 0.1–1000 ppb. The visual detection limitf the assay [0.1 ppb (Fig. S3, strip 5 in supporting information)]ould be defined as the minimum Hg2+ concentration producinghe colour of the test line significantly weaker than that of con-rol line. Although the results of the assay is not accurate thanhat of enzymatic detection (Prakash et al., 2008), liquid chromato-raphic analysis (Dago et al., 2009), fluorescence detection (Wangt al., 2009), colorimetric detection (Takahashi et al., 2009), electri-al detection (Chen et al., 2008a) and mass spectrometry (ICP-MS)Minnich et al., 2008), it shows great promise for simple on-siteisual detection of Hg2+ in water.

.2. Cross-reactivity of the test strip

The cross-reactivity of the test strip with other metal ions Ca2+,o2+, Al3+, Ag+, Cr+, Cr6+, Fe2+, Cd2+, Fe3+, Mg2+, Mn2+, Pb2+ andu2+ at the concentrations from 0.1 to 5000 ppb for each of metal

ons were examined. The results were shown in Table 1. No cross-eactivities were observed with Ca2+, Co2+, Al3+, Cr+, Fe2+, Cd2+, Fe3+,g2+, Mn2+, Pb2+ and Cr6+ up to 5000 ppb concentrations. Only low

ross-reactivities were observed with Cu2+ at the concentration of000 ppb and with Ag+ at the concentration of 1000 ppb. However,his kind of metal ions usually exist in environment at low concen-rations. Therefore the test strip could be used for the detection ofg2+.

.3. Detection of Hg2+ in different spiking water samples

Different water samples were spiked with 0.1, 1.0, 10, 100 and000 ppb of Hg2+ and then analyzed by the test strip (Table 2).he results showed that the different water samples correspondell, and the matrices of the different water samples where Hg2+

ay be found do not interfere with the assay. The detectionimit of the assay is about 1 ppb, close to that of competitiveIA (Marx and Hock, 2000). Although the qualitative test stripssay based on the visual evaluation of results is not accurate

han that of nanocomposite membrane detector assay (Díez-Gilt al., 2008), ELISA (Wylie et al., 1991), ion chromatography (Liu,009), Rhodamine-based ratiometric fluorescence detection (Liut al., 2009), bifunctional fluorescent sensor detection (He et al.,009), fluorescent coumarinylalkyne detection (Lee et al., 2009b),

− − −ne.e.

DNA oligonucleotides and unmodified gold nanoparticles sens-ing system detection (Xu et al., 2009), Electrochemical surfaceplasmon resonance (Panta et al., 2009), oligonucleotide-based flu-orometric detection (Liu, 2008), voltammetric detection (Xu et al.,2008), cationic porphyrin-based self-assembled film (Fang and Liu,2008) and liposome system detection (Yigit et al., 2009), whichrequire expensive equipment and skilled analysts, or are limited inlaboratory scientific research, and the sample preparation is labor-intensive and time consuming. It is easy to perform and could beaccomplished within 15 min without the need of any equipmentand complicated handling procedures. With respect to its overallspeed, convenience and simplicity, this portable device is supe-rior to other methods. Therefore, this test strip could be appliedas screening method for rapid detection of Hg2+ on-site.

4. Conclusion

We have designed a novel probe specific for Hg2+ and developeda one step competitive immunochromatographic assay formatbased on the probe for the detection of Hg2+ in water samples. Toour knowledge, this is the first time in this format for the analysisof Hg2+. The assay is convenient, easy, and can be performed within15 min. The proposed one step assay provides an alternative toolfor rapid, convenient and quantitative detection of Hg2+ on-site.

Acknowledgements

The authors are thankful to the financial support of theNational Nature Science Foundation of China (NSFC, Nos. 60971011,30771657). Talented man support project of Jilin University (No.4305050102J9). Grants of China 985 program and Program forChangjiang Scholars and Innovative Research Team in UniversityIRT0727.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.bios.2010.04.003.

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