Analytica Chimica Acta 827 (2014) 47–53

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Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca

N-Methylimidazolium modified magnetic particles-assisted highly sensitive Escherichia coli detection based on polymerase chain reaction and capillary electrophoresis Manchen Deng, Yu Wang, Li Jia * MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, South China Normal University, Guangzhou 510631, China

H I G H L I G H T S

 MIm-MPs were used as adsorbents for rapid and highly efficient capture of E. coli.  A MIm-MPs-PCR-CE method was developed for sensitive detection of E. coli.  Trace amount of E. coli at 101 cfu mL1 was detected.

G R A P H I C A L A B S T R A C T

?

A R T I C L E I N F O

A B S T R A C T

Article history: Received 14 November 2013 Received in revised form 9 April 2014 Accepted 10 April 2014 Available online 15 April 2014

Effective bacteria detection and quantification are essential prerequisite for the prevention and treatment of infectious diseases. Herein, we report a method for the detection and quantification of Escherichia coli (E. coli). N-Methylimidazolium modified magnetic particles (MIm-MPs) are synthesized successfully and used as an efficient magnetic material for the isolation and concentration of E. coli. The factors including pH of binding buffer, concentration of elution buffer and elution time which may affect the capture and elution efficiencies are optimized. The linear correlation between bacteria concentration and peak area of polymerase chain reaction (PCR) product analyzed by capillary electrophoresis (CE) is determined. Rapid preconcentration of trace amount of E. coli (101 cfu mL1) in large volume of aqueous sample (500 mL) is achieved, and the capture efficiency can reach 99%. The quantification of bacteria in large volume of spiked tap water and mineral water samples is realized. The recoveries for different concentrations of E. coli in tap and mineral water samples are in the range between 83% and 93%. The results demonstrate that this MIm-MPs-PCR-CE method can be applied to detect and quantify bacteria in real samples. ã 2014 Elsevier B.V. All rights reserved.

Keywords: N-Methylimidazolium Magnetic particles Capillary electrophoresis Polymerase chain reaction Escherichia coli

1. Introduction Bacteria are found all over nature and the environment. They are widely existed in soil, nature waters, intestinal tract and skin of human and animals. Most of the bacteria do their duty in the ecological system, and many are closely related to plants or animals in beneficial relations. However, some of the infectious diseases can

* Corresponding author. Tel.: +86 20 85211543; fax: +86 20 85216052. E-mail address: [email protected] (L. Jia). http://dx.doi.org/10.1016/j.aca.2014.04.018 0003-2670/ ã 2014 Elsevier B.V. All rights reserved.

be caused by the inbreak of bacteria into the viscera. Therefore, the development of detection and identification methods for bacteria is necessary in public health, water, and food safety. The conventional plate counting method based on selective growth of the bacteria from a contaminated sample is the most used technique for the detection of bacteria [1]. However, the major defect of the conventional method is excessively timeconsuming. It usually takes a few days to get a result. In order to shorten the detection time for bacteria, the polymerase chain reaction (PCR) based method has been developed for the fast and selective detection of target bacteria [2–4]. In PCR, DNA template

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plays a vital role. In order to obtain a certain concentration of pure DNA template, the amount of bacteria in a sample must reach a certain level. However, the concentration of bacteria in some real samples is far less than the required amount. Thus, the sample containing low concentrations of bacteria needs an incubation step to increase the bacteria concentration. The step is also timeconsuming, which usually takes a few hours to days [5–7]. In recent years, magnetic particles (MPs) receive increasing attention with rapid development in the fields of biotechnology and medicine because of its large specific surface area, strong magnetic property and low toxicity [8–16]. Many kinds of functionalized MPs have been used in drug carrier [8], proteins purification [9,10], metal ions removal [11], triazine herbicides extraction [12], and DNA isolation from biologic samples [13–16]. Poly(1-vinylimidazole)-grafted MPs were synthesized and used to remove Cu2+, Ni2+ and Co2+ from aqueous solutions based on the complexation between poly(1-vinylimidazole) and these metal ions [11]. Graphene-coated MPs were used as adsorbents for magnetic solid phase extraction of triazine herbicides via p-stacking interaction between delocalized p-electron system of graphene and benzenoid compounds [12]. Recently, we prepared N-methylimidazolium modified MPs (MIm-MPs), and they were employed to isolate DNA from genetically modified soybeans by the electrostatic interaction between negatively charged DNA and positively charged MIm-MPs [13]. Some functionalized MPs have been demonstrated exciting and promising applications for capture or removal of bacteria [17,18]. IgG modified MPs can be employed for capturing Staphylococcus saprophyticus and Staphylococcus aureus in aqueous sample solutions by selectively binding to the cell walls of these pathogens containing IgG-binding sites [17]. Amine-functionalized MPs were applied for rapid and efficient capture and removal of pathogens from water, food, and urine samples via electrostatic interactions between the negatively charged bacteria and positively charged amine-functionalized MPs [18]. The capture mechanism of aminefunctionalized MPs for bacteria inspired us to investigate the application of MIm-MPs in bacteria capture as MIm-MPs are positively charged due to the existence of N-methylimidazolium, which were proved by the isolation of negatively charged DNA using MIm-MPs from genetically modified soybeans [13]. Here, we report a method for the concentration, detection and quantification of E. coli in large volume of aqueous solutions by combination of MIm-MPs assisted concentration, PCR and capillary electrophoresis (CE) techniques. The pH of binding buffer, the concentration of elution buffer, and elution time were optimized. The identification and quantification were realized by PCR and CE analysis. 2. Experimental 2.1. Chemicals and reagents Ferric chloride hexahydrate (FeCl36H2O), ammonium hydroxide (NH3H2O, [25%, w/w]), sodium hydroxide (NaOH), tris (hydroxymethyl) amino methane (Tris), ethylenediamine tetraacetic acid (EDTA), boric acid (H3BO3), borax, trisodium citrate dihydrate, and phosphoric acid (H3PO4) were all purchased from Guangzhou Chemical Regent Factory (Guangzhou, China). Tetraethyl orthosilicate (TEOS) was acquired from Acros Organic (New Jersey, USA). N-Methylimidazole was obtained from J&K chemicals (Beijing, China). Methanol, ethanol, and isopropanol (HPLC grade) were purchased from SK chemicals (Ulsan, South Korea). 3Chloropropyltrimethoxysilane was purchased from Aldrich (Saint Louis, USA). Hydroxypropylmethylcellulose (HPMC), the viscosity of which in 2% aqueous solution at 25  C was 250 mPa s, was purchased from Bio Basic Inc. (Toronto, Canada).

TIANamp Bacteria DNA Kit (DP302) was obtained from TianGen Biotech (Beijing, China). The GeneRuler 100-bp DNA ladder was purchased from ChinaGen (Shenzhen, China). The primers used for PCR were synthesized by Shanghai Sangon Biological Engineering & Technology (SSBE, Shanghai, China). Taq DNA polymerase (5 U mL1), 10  PCR buffer (100 mM Tris–HCl [pH 8.3], 500 mM KCl, 15 mM MgCl2), and deoxynucleotide triphosphate (dNTP) mixture (including dATP, dGTP, dCTP, and dTTP, where the concentration of each dNTP was 2.5 mM) were purchased from Takara Biotechnology (Dalian, China). All aqueous solutions were prepared with deionized water (18.2 MV) purified by an Elga water purification system (ELGA, London, UK). 2.2. Synthesis and characterization of MIm-MPs The MIm-MPs were synthesized using our previously developed method [13]. The whole procedure was divided into four steps. Firstly, the preparation of magnetic Fe3O4 particles was based on previously reported solvothermal reduction method [19]. FeCl36H2O (1.35 g) was dissolved in ethylene glycol (40 mL) with the help of ultrasonication. Then, sodium acetate anhydrous (3.6 g) was added to the solution, and the mixture was stirred vigorously for 30 min to form a transparent solution. The solution was transferred into a teflon-sealed stainless-steel autoclave (50 mL) and reacted at 180  C for 10 h. The magnetic Fe3O4 products were washed three times with ethanol and dried at 60  C under nitrogen atmosphere. Secondly, the magnetic Fe3O4 particles were coated with silica [20]. Briefly, Fe3O4 (0.3 g) was dispersed in 40 mL isopropanol under ultrasonication, followed by adding 2 mL H2O and 0.5 mL NH3H2O under vigorous stirring. Then, 0.3 mL TEOS was added to the mixture drop by drop. The final mixture was stirred at room temperature for 8 h. The obtained products were washed three times with ethanol and dried at 60  C under nitrogen atmosphere. Thirdly, the silica coated magnetic particles were functionalized with chloropropyl group. The silica coated magnetic particles (0.4 g) were suspended in 60 mL toluene, followed by addition of 5 mL 3-chloropropyltrimethoxysilane and 0.25 mL triethylamine (as catalyst). The mixture was stirred and refluxed at 90  C for 12 h. Then, the products were washed with 50 mL toluene, 50 mL ethanol, 50 mL ethanol–water mixture (1:1, v/v), and 50 mL water. Finally, the products were dried at 50  C under nitrogen atmosphere. Fourthly, the chloropropyl group modified magnetic particles were functionalized with N-methylimidazolium. In brief, 0.3 g chloropropyl group modified magnetic particles were dispersed in 60 mL toluene. Then, 5 mL N-methylimidazole was added. The suspensions were refluxed at 70  C for 12 h. The resultant products (MIm-MPs) were washed with methanol and water several times and dried at 50  C under nitrogen atmosphere. The TEM images of the MIm-MPs and the conjugates between MIm-MPs and E. coli were carried out on a JEM-2100HR transmission electron microscope (JEOL, Tokyo, Japan). The FT-IR spectra of N-methylimidazole, Fe3O4 particles, SiO2@Fe3O4, and MIm-MPs were recorded on a Tensor 27 FT-IR spectrophotometer (Bruker Corporation, Ettlingen, Germany). The zeta potentials of MIm-MPs at different pH values were measured on a Zetasizer Nano ZS (Malvern, Worcestershire, UK). 2.3. Preparation of bacteria samples Gram negative bacteria E. coli was chosen as a model bacterium. The strain was grown in Luria–Bertani broth (tryptone 10 g L1, yeast extract 5 g L1 and NaCl 10 g L1, pH 7.5) at 37  C for 8 h. The bacteria cells were centrifuged at 7500 rpm for 3 min and then

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washed with borate buffer (50 mM) three times. Different concentrations of bacteria were adjusted using borate buffer, and their optical densities at 600 nm (OD600) were measured on a micro-spectrophotometer (K5600, Beijing Kaiao Technology Development Company, Beijing, China). The bacteria concentration recorded in colony forming units (cfu) was obtained based on the plate counting method. For safety consideration, all the containers were placed in a high-pressure steam sterilization pot at 121  C for 30 min for sterilization before and after use. 2.4. E. coli capture and elution efficiency MIm-MPs (10 mg) were weighed and dispersed in borate buffer (50 mM). Then, the dispersive MPs were added to the freshly prepared bacterial solution whose concentration was adjusted to OD600 value 0.5 using 50 mM borate buffer. The volume of the mixture was 5 mL. The mixture was shaken vigorously for 1 min at room temperature. Then, the MPs–bacteria conjugates were isolated by a magnetic rack (Dynal MPC-S, Invitrogen Corporation, Carlsbad, CA, USA). Supernatants were moved to a new tube by a pipet, and its OD600 value was measured using a microspectrophotometer. The capturing efficiency of MIm-MPs for bacteria can be obtained according to the decrease in the OD600 value after the capture procedure. The effect of pH of binding buffer on the capture of E. coli was evaluated in the range from 3.0 to 9.0. The MPs-bacteria conjugates were washed with 2 mL borate buffer (50 mM) to wipe off unbound bacteria, and allowed to dry at room temperature. Then, 5 mL citrate buffer (pH 7.0) was added. The tube was shaken vigorously for different duration times to desorb the bacteria from MIm-MPs. Then, the magnetic separation was carried out, and the OD600 value of the supernatant was measured. The elution efficiency was derived from the difference between the amount of bacteria in the supernatant and the amount of bacteria adsorbed on MIm-MPs. The effect of the concentration of elution buffer (citrate buffer) on the elution was evaluated in the range from 0.1 to 1.5 M. The influence of the elution time on the desorption of bacteria from MIm-MPs was optimized in the range of 5–90 min.

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25  C. The background solution consisted of 1% (w/v) HPMC, 2 mM EDTA, and 20 mM phosphoric acid, whose pH was adjusted to 7.3 using Tris solution. Samples were injected electrokinetically by 10 kV for 10 s at the cathodic end and separated by applying 10 kV. The detection wavelength was set at 260 nm [21]. Before first use, a bare uncoated capillary was preconditioned by flushing sequentially at 138 kPa with 1.0 M NaOH (15 min), methanol (10 min), deionized water (10 min), and the background solution (30 min). Between each run, the capillary was equilibrated by flushing at 138 kPa with methanol for 3 min, 0.1 M HCl for 3 min, and the background buffer for 3 min in sequence to ensure the reproducibility. All solutions used in electrophoresis experiments were deaerated by ultrasonication prior to use. 2.7. Quantification of E. coli from real samples using MIm-MPs-PCR-CE method MIm-MPs were used to concentrate E. coli from large volume of aqueous solutions, as shown in Fig. 1. The mineral water used for this study was purchased from a local market. The tap water was collected from our lab. MIm-MPs (50 mg) were dispersed in a conical flask containing 500 mL sample solution with the help of ultrasonication for 30 min. The solution pH was adjusted to 7.0 with 1.0 mol L1 NaOH or 1.0 mol L1 HCl. The conical flask was vigorously shaken for 10 min to ensure the interaction of MIm-MPs with E. coli. The MIm-MPs-bacteria conjugates were isolated from aqueous samples by a magnet and transferred into a 15 mL tube. Then 10 mL 0.2 M citrate buffer (pH 7.0) was added, and the mixture was vigorously shaken for 60 min to elute E. coli from the particles. An external magnet was employed for magnetic separation. And the eluted bacteria were transferred to a 1.5 mL tube. The bacteria DNA kit was used for the isolation of genomic DNA from the eluted E. coli. Then, the genomic DNA was taken for PCR, followed by CE analysis. The uncaptured bacteria concentration in the supernatant after magnetic separation was measured using plate counting method. 3. Results and discussion

2.5. PCR method

3.1. Characterization of MIm-MPs

The isolation of E. coli DNA was completed by using TIANamp Bacteria DNA Kit. PCR amplification was carried out in a solution containing 1  PCR buffer, 0.2 mM of each dNTP, 0.4 mM of each primer, 1.5 mM MgCl2, 0.2 U mL1 Taq DNA polymerase, and 5 mL DNA template. The total reaction volume was 25 mL. The 246 bp fragment was amplified using the follow primers: forward, 50 GAGCGCAACCCTTATCCTTTG-30 , and reverse, 50 -TACTAGCGATTCCGACTTCATGG-30 . The PCR amplification was performed on a gradient thermocycler (A200, Longgene Scientific Instrument Company, Hangzhou, China) with the following protocol: an initial denaturation at 94  C for 5 min, followed by 35 cycles of denaturation at 94  C for 1 min, annealing at 50  C for 45 s, and extension at 72  C for 1 min with a final extension at 72  C for 10 min. The PCR products were analyzed by CE.

The FT-IR spectra of Fe3O4 (a), SiO2@Fe3O4 (b), MIm-MPs (c), and N-methylimidazole (d) are shown in Fig. 2. The peaks at 593 cm1 in curves a–c were related to the Fe O Fe vibration of magnetite. In curves b and c, the strongly absorbing region of 1110–1000 cm1 resulted from the SiO Si stretching vibration on the surface of MPs. The broad band around the region 3300–3500 cm1 was attributed to the stretching vibration of –OH groups of silica. In curve c, the emergence of the new adsorption peaks at 2852 and 2921 cm1 was observed, which was assigned to the C H stretching vibrations in the N-methylimidazole units. The result confirmed that N-methylimidazole has been successfully bonded to the surface of the SiO2@Fe3O4 particles. The zeta potentials of MIm-MPs at different pH values were investigated, as shown in Fig. 3. The zeta potential of MIm-MPs decreased with the increase of the pH values from 2 to 11 except for pH 6–8. And the isoelectric point (pI) of MIm-MPs was calculated to be about 8.7. When the pH was below 8.7, MIm-MPs took positive charges due to the existence of N-methylimidazolium on MIm-MPs. The results implied that the positively charged MImMPs can be used to adsorb negatively charged bacteria through electrostatic interaction at a suitable pH. The particle size and morphology information of MIm-MPs and the conjugates of the MIm-MPs and bacteria were studied by TEM images, as shown in Fig. 4A and B. The TEM images of MIm-MPs demonstrated that MIm-MPs were spherical particles. And the

2.6. CE procedure All capillary electrophoresis experiments were performed on a PACE-MDQ Beckman P/ACETM MDQ CE instrument (Beckman Coulter, Fullerton, CA, USA) equipped with a photodiode array detection UV detector. Data acquisition and analysis were performed with 32 Karat software (Beckman Coulter, Fullerton, CA, USA). Separations were carried out in a fused-silica capillary (58 cm [effective length 48 cm]  75 mm i.d.) (Hebei Yongnian Ruipu Chromatogram Equipment Company, Handan, China) at

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Fig. 1. Illustration of the bacteria capture by MIm-MPs and elution procedure.

TEM images of MPs-E. coli conjugates demonstrated that MIm-MPs were combined with E. coli successfully, which indicated that MImMPs were capable of concentrating E. coli from aqueous solutions. 3.2. Correlation of bacteria concentration and OD600 value The correlation of bacteria concentration and OD600 value was determined based on the plate counting method, as shown in Fig. S1. After least square fitting of the plot C (bacteria concentration in cfu mL1) versus OD600, the equation, C = 1.15  108OD600 – 1.05  105, was obtained (r = 0.9941), which indicated that the bacteria concentration has good linear relationship with OD600 value. And when the OD600 value was 1, the bacteria concentration was equivalent to 1.15  108 cfu mL1. Based on the correlation of bacteria concentration and OD600 value, the bacteria concentration was determined according the OD600 value in further experiments. 3.3. Optimization of capture and elution conditions The bacteria capture and elution efficiencies of MIm-MPs were evaluated using E. coli as a model bacterium. The factors

including pH of binding buffer, concentration of elution buffer, and eluting time were optimized. Firstly, the effect of the pH of binding buffer (50 mM borate) in the range from 3.0 to 9.0 on the bacteria capture by MIm-MPs was investigated, as shown in Fig. S2A. The capture efficiencies were almost close to 100% and remained constant in the pH range from 3.0 to 7.0. Beyond pH 7.0, the capture efficiency sharply decreased. The MIm-MPs are positively charged below pH 8.7 as the pI of MIm-MPs was about 8.7 (as shown in Fig. 3). The pI of E. coli is about 4.4 [22]. E. coli cell surfaces are negatively charged by virtue of the ionized phosphoryl and carboxylate substituents on outer cell envelope macromolecules [23] when pH was above 4.4. Therefore, the capture of E. coli by MIm-MPs in the pH range from 5.0 to 7.0 mainly resulted from the electrostatic interactions between the positively charged MIm-MPs and the negatively charged E. coli. It is surprising in our study that the capture efficiency of MIm-MPs for E. coli was also close to 100% when the pH of binding buffer was below 4.4 (3.0 and 4.0). Silica coated MPs were used as a control to test their capture ability for E. coli. The optical density of supernatant after magnetic capture by silica coated MPs was almost the same as that of E. coli solution without MPs, indicating that silica coated MPs were nearly incapable of

Fig. 2. FT-IR spectra of Fe3O4 (a); SiO2@Fe3O4 (b); MIm-MPs (c); and N-methylimidazole (d).

M. Deng et al. / Analytica Chimica Acta 827 (2014) 47–53

Fig. 3. Zeta potentials of MIm-MPs at different pH values. pH 8.7 is the isoelectric point of MIm-MPs.

capturing E. coli. The zeta potential of silica coated MPs in 50 mM borate buffer (pH 7.0) was measured to be 40.8 mV. The electrostatic repulsion between anionic silica coated MPs and negatively charged E. coli led to the incapability of silica coated MPs to capture E. coli. The results confirmed that the capture of E. coli by MIm-MPs was through electrostatic attractions between the positively charged MIm-MPs and the negatively charged E. coli. Considering the stability of E. coli and the capture efficiency of MIm-MPs for E. coli, pH 7.0 was used for subsequent adsorption assays.

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Secondly, the type and the concentration of elution buffer were investigated. Several buffers (5 mL) including citric acid, citrate, phosphate, and carbonate buffer solutions were used to elute E. coli from MIm-MPs-E. coli conjugates. The desorption ability of citrate buffer was found to be superior and was chosen as the elution buffer. The concentration of citrate buffer (pH 7.0) in the range from 0.1 to 1.5 M was optimized, as shown in Fig. S2B. When the concentration of citrate was below 0.2 M, the elution efficiency increased with the increase of the citrate concentration. The elution efficiency was found to be 96% when the citrate concentration was 0.2 M. Using citrate ions to desorb the bacteria from the particles should be based on the ion-exchange mechanism as the electrostatic interactions played the major roles for the capture of bacteria by MIm-MPs. The negatively charged citrate ions have stronger electrostatic interaction with MIm-MPs than bacteria, and thus, can compete with and replace bacteria from the MPs-bacteria conjugates. Beyond 0.2 M, the elution efficiency had a drop with the increase of the citrate concentration, which is possibly caused by the death of E. coli in high ionic strength of the solutions [24]. Thus, the citrate buffer (0.2 M, pH 7.0) was used for subsequent experiments. Thirdly, the elution time was optimized when 0.2 M citrate buffer was used as the elution buffer. As shown in Fig. S2C, the elution efficiency increased with the increase of the elution time before 60 min. Further prolonging the elution time led to the decrease of the elution efficiency. At elution time 60 min, the elution efficiency was 96%. Elution time 60 min was used for further experiments. The binding capacity of the MIm-MPs for E. coli was determined. The MIm-MPs (0.5 mg) were used as the adsorbents, and the solutions containing different concentrations of E. coli in the same volume of binding buffer (0.5 mL) were extracted. After magnetic extraction, the amount of E. coli remaining in the supernatant was determined. As shown in Fig. 5, the E. coli adsorption amount by MIm-MPs was linear in the range of 1.2  107–6.7  107, beyond which the adsorption amount reached a plateaus. The binding capacity of the MIm-MPs for E. coli was calculated to be 1.3  108 cfu mg1. 3.4. Correlation of bacteria concentration and peak area of PCR product analyzed by CE In this study, PCR product was not only used for the identification of E. coli but also for the quantification of E. coli.

Fig. 4. HRTEM images of MIm-MPs (A) and MIm-MPs–E. coli conjugates (B).

Fig. 5. Binding capacity of MIm-MPs for E. coli. The volume of different concentration of E. coli, 0.5 mL; the amount of MIm-MPs, 0.5 mg, the incubation time, 1 min. The binding capacity of the MIm-MPs for E. coli was calculated to be 1.3  108 cfu mg1.

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PCR product was analyzed by a sieving CE [21]. Therefore, the correlation of E. coli concentration and peak area of PCR product analyzed by CE is necessary. Five different concentrations of E. coli were used to isolate DNA, followed by PCR and CE analysis. As shown in Fig. S3, the correlation between E. coli concentration and the peak area of PCR product can be represented by an equation, C = 0.008 A  2.85 (r = 0.9906), where C is E. coli concentration and A is the peak area of PCR product. The equation demonstrated that the bacteria concentration has linear relationship with the peak area of PCR product analyzed by CE. Fig. 6 showed the electropherograms of the standard GeneRuler 100-bp DNA ladder, the PCR product of 246 bp with E. coli DNA template, and the blank without E. coli DNA template. In the electropherogram of the PCR product with E. coli DNA template, one peak appeared between the peaks of 200 and 300 bp fragments. The size of the PCR product was determined using standard GeneRuler 100-bp DNA. Because the peak of PCR product of E. coli is located between the peaks of 200 and 300 bp fragments, the data of the migration times (tm) and the size corresponding to the standard DNA fragments from 100 to 500 bp were employed to determine the size of PCR product. After least square fitting of the plot log (bp) versus 1/tm (Fig. S4), the equation, log (bp) = 3.90  36.7/tm was obtained (r = 0.9997), where bp is the number of base pair of DNA, tm is the migration time of analyte. This equation was used to determine the number of base pair of the PCR product of E. coli based on its migration time. The calculated value was 244 bp, which is basically in agreement with the real value 246 bp. The results indicated that the target PCR product was amplified successfully. 3.5. Quantification of E. coli in spiked real samples using MIm-MPsPCR-CE method In order to enhance the detection sensitivity of E. coli, the adsorption of E. coli by MIm-MPs from large volume of sample solutions was combined with the PCR-CE method. Tap water and mineral water were chosen to demonstrate the feasibility of the developed MIm-MPs-PCR-CE method. E. coli was not detected in the tap water and mineral water using the plate counting method. Thus, 500 mL spiked water sample containing 101 cfu mL1 E. coli was used as the test sample solution. For large volume of sample, the effects of the amount of MIm-MPs and the binding time on the adsorption efficiency were investigated. The results demonstrated that the adsorption efficiencies of MIm-MPs for E. coli from tap and mineral water samples were larger than 99% when 50 mg MImMPs was used and the adsorption time was 10 min. The adsorbed E. coli by MIm-MPs was eluted using 0.2 M citrate buffer, followed by

Table 1 Recovery results for different concentrations of E. coli in water samples. Sample

Added E. coli (cfu mL1)

Found E. coli (cfu mL1)

Recovery (%)

RSD (n = 3) (%)

Tap water

57 80 103

50 75 95

88 93 92

7.9 5.2 5.6

Mineral water

57 80 103

47 73 93

83 91 90

6.7 7.3 6.0

the isolation of genomic DNA, PCR, and CE analysis. The recovery study of the method was performed at different concentrations of E. coli in water samples, as shown in Table 1. The recoveries for tap and mineral water samples were in the range between 83% and 93% with relative standard deviations (RSDs) less than 7.9%. The results indicated that using MIm-MPs as adsorbents for capture E. coli can effectively enhance the detection sensitivity, and the developed MIm-MPs-PCR-CE method is suitable for rapid and sensitive detection and quantification of low levels of bacteria in aqueous solutions. 4. Conclusion We developed a MIm-MPs-PCR-CE method for the rapid and sensitive detection and quantification of E. coli. The MIm-MPs were used as adsorbents for the preconcentration of trace amounts of E. coli from large volumes of spiked real samples. E. coli was captured by MIm-MPs based on the electrostatic interactions and desorbed from the particles by citrate ions based on the ion-exchange mechanism. The identification and quantification of E. coli were completed by PCR and CE analysis. Through combining the preconcentration of E. coli by MIm-MPs from large volume of sample solutions with the PCR-CE method, the order of 101 cfu mL1 of E. coli in samples can be detected. In comparison with other functionalized MPs which aimed the capture of bacteria [17,18,25,26], the developed MIm-MPs-PCR-CE method showed the superiority in the capture time and efficiency, and the detection sensitivity. The developed method was successfully applied for the rapid detection of low levels of E. coli in the spiked tap water and mineral water samples. Acknowledgements We are grateful to the financial support of the National Natural Science Foundation of China (21075044 and 21175048) and we thank Congjie You in South China Normal University for providing the strain of E. coli. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aca.2014.04.018. References

Fig. 6. Analysis of PCR product by CE.

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N-Methylimidazolium modified magnetic particles-assisted highly sensitive Escherichia coli detection based on polymerase chain reaction and capillary electrophoresis.

Effective bacteria detection and quantification are essential prerequisite for the prevention and treatment of infectious diseases. Herein, we report ...
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