Label-Free Electrical Immunosensor for Highly Sensitive and Specific

Article pubs.acs.org/est

Label-Free Electrical Immunosensor for Highly Sensitive and Specific Detection of Microcystin-LR in Water Samples Feng Tan,*,†,‡ Nuvia Maria Saucedo,† Pankaj Ramnani,† and Ashok Mulchandani*,† †

Department of Chemical and Environmental Engineering, University of California, Riverside, California 92521, United States Key Laboratory of Industrial Ecology and Environmental Engineering (MOE), School of Environmental Science and Technology, Dalian University of Technology, Dalian 116024, China

‡

ABSTRACT: Microcystin-LR (MCLR) is one of the most commonly detected and toxic cyclic heptapeptide cyanotoxins released by cyanobacterial blooms in surface waters, for which sensitive and specific detection methods are necessary to carry out its recognition and quantification. Here, we present a single-walled carbon nanotube (SWCNTs)-based label-free chemiresistive immunosensor for highly sensitive and specific detection of MCLR in different source waters. MCLR was initially immobilized on SWCNTs modified interdigitated electrode, followed by incubation with monoclonal anti-MCLR antibody. The competitive binding of MCLR in sample solutions induced departure of the antibody from the antibody−antigen complexes formed on SWCNTs, resulting in change in the conductivity between source and drain of the sensor. The displacement assay greatly improved the sensitivity of the sensor compared with direct immunoassay on the same device. The immunosensor exhibited a wide linear response to log value of MCLR concentration ranging from 1 to 1000 ng/L, with a detection limit of 0.6 ng/L. This method showed good reproducibility, stability and recovery. The proposed method provides a powerful tool for rapid and sensitive monitoring of MCLR in environmental samples.

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INTRODUCTION Cyanobacterial bloom has been an increasing threat to ecological environments and public health worldwide due to the release of various cyanotoxins into water sources. Microcystin (MC) is one of the most commonly detected cyclic heptapeptide cyanotoxins, which can cause acute liver damage to mammals at high doses and render tumor promotion for low level chronic exposure by inhibiting the activity of protein phosphatases 1 and 2A and thus destroying the dynamic balance of protein phosphorylation in the body.1,2 To date, more than 90 MCs have been identified from cyanobacteria genera. Among them, microcystin-LR (MCLR), containing five nonproteinogens and two substitutions of leucine (L) and arginine (R) at positions 2 and 4, is the most toxic congener with a LD50 of 43 μg/kg.3 Because of its acute toxicity, in 1998 the World Health Organization (WHO) set a guideline value of 1 μg/L for MCLR in drinking water.4 Hence, there is a great need to develop reliable analytical methods for ultrasensitive detection of low concentration MCLR in the fields of environmental monitoring, food safety and toxicity assessment. © XXXX American Chemical Society

The usual techniques for MCLR detection mainly include high-performance liquid chromatography (HPLC) combined with either an ultraviolet−visible absorbance detector or a tandem mass spectrometer (MS/MS),5,6 and protein phosphatase inhibition (PPI) assays based on radioisotopic, colorimetric, and electrochemical measurements.7,8 HPLC methods are reliable and widely accepted, however, they require relatively expensive equipment, high cost, and are timeconsuming and not competent for on-site detection of MCLR in emergent pollution events. PPI methods based on colorimetric and electrochemical detections have advantages of simplicity, low cost and sufficient sensitivity; however, multiple MC variants show different inhibitory potencies, resulting in false positive results.9 Additionally, the cyanobacterial sample itself may contain phosphatase activity, resulting in large quantification deviations.10 Received: April 2, 2015 Revised: June 24, 2015 Accepted: June 29, 2015

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DOI: 10.1021/acs.est.5b01674 Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Article

Environmental Science & Technology

MCLR to the anti-MCLR antibody immobilized on SWCNTs, which constituted the sensing channel. The developed sensor showed good precision, reproducibility and stability, and could be successfully applied in the detection of MCLR in tap, lake, and river waters with satisfactory recoveries. The proposed strategy provides a powerful tool for rapid and sensitive analysis of MCLR in environmental samples.

Immunoassays for MCLR detection based on monoclonal or polyclonal antibodies have been extensively studied due to their high sensitivity and specific molecular recognition, without the need for complicated sample pretreatment procedures. Examples include, fluorescent immunoassay, enzyme-linked immunosorbent assay (ELISA) and various immunosensors based on colorimetric,11 fluorescent,12,13 chemiluminescent,14,15 and electrochemical16,17 signals. The immunoassays are usually carried out by either direct or indirect/competitive format, which involve chemical labeling or modification of small antigen MCLR or antibody with proteins,18,19 catalytic enzymes,20−22 or fluorescent dyes.23 Furthermore, nanomaterials, such as carbon nanohorn,16 carbon nanotube,20 graphene,17 TiO2 nanotube,24 Au nanoparticles,11 Ag nanoparticles,25 and quantum dot (QD),26 magnetic nanoparticles,27,28 mesoporous PtRu alloy,29 were conjugated with antibody as signal transducer and enhancement factors to improve the response speed and sensitivity due to large surface area, and excellent structural, electronic, and optical characteristics. These sensors enabled the quantification of MCLR from μg/L to ng/L levels in tap and ground waters. However, the preparation and separation of the labeled small antigen MCLR and antibody are very complicated, resulting in the increase of analytical time, cost, and unforeseen errors. In addition, labeling may influence the affinity interaction between antibody and small antigen MCLR. Label-free immunosensors for MCLR detection have been gaining interest because of the elimination of labels, i.e. simplicity, without sacrificing sensitivity. Examples include, immunosensors based on piezoelectric,30 fluorescence,12 electrochemical,9,31 photoelectrochemical,24 capacitive,25,32 impedimetric,33,34 and surface plasmon resonance (SPR)35 signals with μg/L to ng/L, and in one case pg/L, levels of sensitivity. Despite these progresses, novel sensors with simpler operation, lower cost, faster response, and more reliable analysis are continually needed. Recently, one-dimensional (1-D) nanostructure-based fieldeffect transistors (FET)/chemiresistors have been rapidly gaining attention as powerful transducers in biosensors,36,37 which can offer promising advantages in terms of feasible miniaturization, cost-effectiveness, sensitivity, and analysis time. Of several different 1-D nanomaterials, single-walled carbon nanotube (SWCNTs) have emerged as a promising material for the development of FET/chemiresistive biosensors due to the extreme sensitivity and response speed toward the change in the surface microenvironment of the transfer channels derived from the adsorption/modification of extraneous molecules.38,39 In addition, facile surface modifications can be achieved on carbon nanotubes, giving many possibilities for noncovalent and covalent immobilization of small molecules and large biological molecules.40 In general, adsorption of large biological molecules containing charges might result in a larger perturbation in the transfer of electrons within carbon nanotubes by the effects of electrostatic gating, changes in gate coupling, and carrier mobility changes than compared with the adsorption of small molecules and neutral species,41,42 thus resulting in higher sensitivity. In fact, single-molecule detection of proteins and DNA hybridization has been reported by carbon nanotube-based FET biosensors.43,44 In the present work, we developed a chemiresistive immunosensor based on SWCNTs for label-free detection of MCLR in different source waters. High sensitivity and specificity was obtained by the competitive binding of free

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MATERIALS AND METHODS Materials and Apparatus. 3-Aminopropyltriethoxysilane (APTES) was purchased from Sigma-Aldrich (USA). MCLR and microcystin-RR (MCRR) were purchased from Life Science Inc. (USA) and microcystin-LW (MCLW) was purchased from Merck Inc. (Germany). Monoclonal mouse anti-MCLR antibody (IgG1, B764M) was purchased from GeneTex Inc. Ninety five percent semiconducting preseparated SWCNTs solution (0.01 mg/mL) was obtained from NanoIntegris Inc. 1-Pyrenebutanoic acid succinimidyl ester (PBASE) was purchased from Invitrogen Inc. A CHI660 electrochemical workstation was used to obtain current−voltage curves and a scanning electron microscope (Leo 1550) was used to examine morphology of the chemiresistive immunosensor. Fabrication of SWCNTs Chemiresistive Immunosensor. Sensing devices were microfabricated on a highly doped ptype silicon substrate (Ultrasil Corp., 4″ Prime Silicon Wafer, P/Boron doped, ⟨100⟩ orientated, 525 ± 25 μm thick, 0.01− 0.02 Ohm-cm) with 300 nm SiO2 insulating/dielectric layer by standard lithographic patterning. Interdigitated electrodes were written on the substrate by photolithography, followed by the deposition of a 20 nm Cr layer and a 180 nm Au layer by ebeam evaporation. Interdigitated electrodes were cleaned sequentially with acetone and piranha solution (a mixture of sulfuric acid and 30% hydrogen peroxide (3:1, v/v)) followed by drying with N2 after each step. The chip was incubated with APTES for 30 min to assist SWCNT immobilization after which the electrodes were rapidly washed with plenty of deionized water and dried with N2. Subsequently, the interdigitated electrodes were incubated with 5 μL SWCNT suspension for 1 h under high humidity conditions to prevent fast evaporation of the SWCNT solution, washed with deionized water and annealed at 250 °C for 1 h in ambient air. The electrodes with assembled SWCNT were incubated with 6 mM 1-pyrenebutanoic acid succinimidyl ester (PBASE) in dry dimethylformamide (DMF) for 30 min, washed with DMF and phosphate buffer (PB, 10 mM, pH 7.4), and then incubated with 1 μg/mL MCLR for 1 h at room temperature. The electrodes were then incubated with 20 mM ethanolamine (EA) for 1 h to react residual ester groups of PBASE followed by 0.1% Tween-20 for 1 h to block any naked/bare sites on the SWCNTs to prevent any nonspecific adsorption, and then rinsed thoroughly with PB solution. Next, the electrodes were incubated with 10 μg/mL of monoclonal mouse anti-MCLR antibody for 1 h and rinsed with copious amount of PB solution to remove excess antibody. The as-prepared immunosensors were maintained in PB solution and refrigerated at 4 °C. Sensing Procedure of Chemiresistive Immunosensor. Sensing procedure consisted of measuring the initial resistance (R0) of the immunosensor (determined from the slopes of I−V curves between +0.1 V and −0.1 V) followed by incubation for 1 h at room temperature with 20 μL of different concentrations of standard MCLR, rinsing 10 times with PB solution (10 mM, pH 7.4) and measuring the resistance (R) again. Relative resistance changes (R-R0)/R0 were plotted as a function of log B


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antibody (B764M) is ∼146 kDa, a displacement or competitive binding immunoassay was used. Scheme 1 shows the schematic

value of MCLR concentration to evaluate the sensor performance characteristics. Analysis of Water Samples. Water samples included tap, lake and river water, which were collected from our laboratory, Perris Lake (Perris, CA) and the Santa Ana River (Riverside, CA) respectively. The water samples were centrifuged at 10 000 rpm for 30 min to remove large suspended substances and then spiked with MCLR standard solution, prepared in 10 mM PB solution, to achieve final concentrations of 10 ng/L and 100 ng/L.

Scheme 1. Schematic Diagram of the Preparation and Detection Procedures of MCLR Chemiresistive Immunosensor

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RESULTS AND DISCUSSION Sensing device was constructed by dropping SWCNTs solution onto the APTES-functionalized SiO2 surface and gold interdigitated electrode, with 3 μm gap between fingers, acting as source and drain of the chemiresistive sensor. This simple drop casting resulted in a uniform and dense carbon nanotube network as the sensing channel in the gap, as shown in Figure 1(b). The starting resistance of the carbon nanotube channels illustration of the chemiresistive biosensor fabrication/sensing steps. The carbon nanotube sensing channel was noncovalently modified with the linker PBASE through π−π interactions between SWCNTs and pyrene. MCLR was immobilized covalently by nucleopholic substitution of the succinimidyl ester groups of PBASE with the amine groups present in MCLR. The unreacted succinimidyl ester groups of PBASE were neutralized with ethanolamine (EA) and the unfunctionalized SWCNTs were blocked with Tween-20 to prevent any nonspecific adsorption.47 Each step of device fabrication and surface modification was verified by monitoring the change in the drain current (IDS) of the channel as a function of the drain voltage (VDS), as shown in Figure 2(a). Increase in the resistance of the conducting channel was noticed for successive modification steps. The resistance increases were in accordance with the literature and was a result of π−π stacking, electron donation and/or scattering potential generated by the immobilization of the different molecules on SWCNTs and thereby decreasing hole mobility.48 When the device was incubated with the anti-MCLR antibody, the resistance of the channel increased ∼2.9 times compared with that after Tween-20 blocking, as shown in Figure 2(b). This great increase in the resistance was attributed to the specific antibody−antigen interaction between the antiMCLR antibody and the MCLR immobilized on SWCNTs, inducing electrostatic gating effect or charge-transfer in the channel upon the affinity binding of the anti-MCLR antibody.49 When the channel was further incubated with 10 ng/L MCLR solution, the resistance of the channel showed a decrease of ∼28% compared with that before the incubation. This decrease was attributed to the competitive binding of the free MCLR to the anti-MCLR antibody immobilized on SWCNTs by antibody−antigen interaction, resulting in release of the antibody from the antibody-MCLR complexes on the SWCNTs sensing channel. Figure 2(b) shows relative average resistance change (R-R0)/R0 after each modification/sensing step of a device with five effective sensing channels. It can be seen that the (R-R0)/R0 value had a great increase with Ab and a definite decrease with 10 ng/L MCLR (p < 0.05). To confirm the competitive binding of MCLR to the antiMCLR antibody immobilized on SWCNTs, a control experiment was carried out with a device where the same modification steps were used except for the initial MCLR

Figure 1. (a) Optical microscope image of microfabricated chip with five pairs of interdigitated electrodes. Each electrode constitutes a sensing channel and has 20 fingers (10 pairs) that are 200 μm long, 5 μm wide and separated by a 3 μm gap. (b) Scanning electron microscope (SEM) image of SWCNTs-modified interdigitated electrode.

between source and drain was 2−5 kΩ. Annealing further improved the contact between carbon nanotubes and gold electrodes, resulting in 6−10 times decrease in the resistance of the channels. For FET/chemiresistive biosensors, biological molecules with large molecular mass and multiple charges such as proteins and DNA could generally result in larger changes in the conductivity of the transfer channels as compared with small molecules within Debye length.45,46 In the present work, considering that the molecular mass of MCLR is only ∼1 kDa while the molecular mass of the monoclonal anti-MCLR C


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Figure 3. Relative resistance change of the device in the fabrication process for control experiment (filled) and displacement assay (hatched), where the PBASE-functionalized electrodes after Tween20 blocking were incubated with 10 μg/mL anti-MCLR antibody and then 10 ng/L MCLR; (hatched) displacement assay, where the PBASE-functionalized electrodes were incubated with 1 μg/mL MCLR and blocked with Tween-20, then incubated with 10 μg/mL anti-MCLR antibody and 10 ng/L MCLR. R0 is the resistance of the device after blocking with Tween-20 for 10 μg/mL Ab column and the resistance of the device after incubation with the anti-MCLR antibody for 10 ng/L MCLR column, respectively, and R is the resistance of the device after incubation with 10 μg/mL anti-MCLR antibody or 10 ng/ L MCLR for the two columns.

antibody−antigen binding interaction, which may induce conformation changes of the anti-MCLR antibody, occurring more adjacent to the SWCNTs surface in the case of displacement binding than compared with the direct immobilization of the antibody, resulting in a larger perturbation in the conductivity of the sensing channel in the former case. When the anti-MCLR antibody-functionalized device was incubated with 100 ng/L MCLR, the device produced only a 7.8% increase in resistance, which was 7-fold smaller than the response (−54.7%) for the same concentration MCLR and even smaller than that for 1 ng/L MCLR (−14.3%) in the displacement assay with chemiresistive biosensor. The results showed that the displacement method achieved higher sensitivity compared with the direct immunoassay with the sensing device. The analytical characteristics, dynamic range, sensitivity and limit of detection, of the chemiresistive immunosensor by displacement assay was investigated by the incubation with various concentrations of MCLR solutions. Figure 4 shows the

Figure 2. (a) IDS−VDS characteristics of chemiresistive immunosensor at different stages of fabrication and upon exposure of 10 ng/L MCLR (1) bare SWCNTs; (2) SWCNTs functionalized with PBASE; (3) after MCLR immobilization; (4) after blocking with EA and Tween 20; (5) after immobilization with 10 μg/mL anti-MCLR antibody; (6) after incubation with 10 ng/L MCLR for 1 h at room temperature. (b) Relative average resistance change (R-R0)/R0 of the five chemiresistive immunosensor after each modifications, where R0 is the initial resistance of the bare SWCNTs channel, and R is the resistance of the channel after each modification (p < 0.05 at 95% confidence interval).

immobilization on SWCNTs. Results showed that the antiMCLR antibody incubation produced a small change in resistance of the device compared with the competitive binding assay (Figure 3), confirming that the anti-MCLR antibody did not attach on the SWCNTs sensing channel in absence of the initial immobilized MCLR. The small response might be attributed to nonspecific adsorption of the antibody to SWCNTs even with Tween-20 blocking. Further, as shown in Figure 3, further incubation of the device with 10 ng/L of MCLR produced an insignificant change in resistance of the device, which might be attributed to the specific interaction between free MCLR and the few antibody on SWCNTs deriving from the nonspecific adsorption. These results confirmed the effectiveness of the proposed displacement sensing method for the detection of MCLR. To show the sensitivity improvement by the present displacement assay in chemiresistive biosensor, we carried out a direct immunoassay with the device, in which the anti-MCLR antibody was initially immobilized on the carbon nanotubes sensing channel through PBASE, followed by neutralization with EA and blocking with Tween-20. The immobilization of the anti-MCLR antibody increased resistance of the device by just ∼10%, which was unexpected (data not shown). This change is small compared with that produced by the affinity binding of the anti-MCLR antibody with MCLR as above (see Figure 2b). This difference might be attributed to the

Figure 4. Chemiresistive immunosensor calibration curve for MCLR. The data points are averages of measurements from nine independent sensors and error bars represent ±1 standard deviation. R0 is the resistance of the SWCNTs channels after incubation with 10 μg/mL Ab and R is the resistance of the immunosensor after incubation different concentration MCLR. D


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Environmental Science & Technology Table 1. Comparison of Various Immunosensors for the Detection of MCLR in Waters measurement signals

analysis mode

competitor

recognition elements

LOD

ref

electrochemical electrochemical electrochemical electrochemical electrochemical electrochemical electrochemical electrochemical electrochemical electrochemical fluorescence fluorescence fluorescence surface-enhanced fluorescence resonant frequency colorimetric SPR chemiluminescence photoelectrochemical electrochemical and colorimetric resistance

indirect indirect indirect direct indirect indirect direct direct direct indirect direct direct indirect indirect direct indirect direct indirect direct indirect direct

MCLR-carbon nanohorns antibody-membrane MCLR MCLR HRP-MCLR SiO2/HRP/antibody MCLR MCLR PtRu-antibody glucose oxidase labeled MCLR MCLR-DNA MCLR-DNA-Au MCLR-OVA Cy5 goat antirabbit antibody antibody MCLR-OVA MCLR MCLR-OVA MCLR MCLR-HRP MCLR

HRP labeled antibody HRP labeled MCLR quantum dot/antibody antibody Au-antibody antibody antibody antibody antibody antibody antibody antibody antibody antibody antibody antibody antibody antibody antibody antibody antibody

30 ng/L 20 ng/L 99 ng/L 37 fg/L 2 ng/L 4 ng/L 7 pg/L 20 ng/L 9.6 ng/L 100 ng/L 140 ng/L 500 ng/L 30 ng/L 7 ng/L 1 ng/L 0.05 μg/L 73 ± 8 ng/L 32 ng/L 55 ng/L 400 ng/L 0.6 ng/L

16 21 26 17 20 28 25 50 29 51 12 52 53 54 30 11 35 14 55 27 Present study

thus sufficient for practical applications. This enhancement in sensitivity is attributed to the extremely high sensitivity of SWCNTs-based chemiresistive transduction combined with the displacement assay principle. Selectivity of Chemiresistive Immunosensor. To examine the selectivity of the label-free chemiresistive immunosensor, two other MCs (MCRR and MCLW) with similar molecular structure as MCLR were incubated with the sensing device, respectively. MCRR has an arginine at the 2position instead of a leucine as in MCLR. Results showed that the response produced by the same concentration of MCRR is less than 10% of that produced by MCLR (Figure 5). As for MCLW, it has a tryptophan at the 4-position instead of an arginine as in MCLR, and was almost not recognized by the sensor due to a large difference in molecular structure at 4position. The results were in line with previous reports,12 which demonstrated that the monoclonal antibody exhibited high

normalized response (R-R0)/R0 of the label-free chemiresistive immunosensor as a function of log value of MCLR concentration, where R0 is the resistance after incubation with anti-MCLR antibody and R is the resistance after incubation with MCLR. As shown in the Figure, the sensor response was linear over the range of 1 ng/L to 1000 ng/L and a linear regression equation of y = −0.175x − 1.72 (R = 0.9925) was obtained. The detection limit (LOD), calculated based on the equation LOD = 3 x standard deviation of blank/ slope of calibration plot was 0.6 ng/L. Table 1 shows the analytical performance of previous immunosensors found in literature for the detection of MCLR. The LOD for our scheme of detection is lower than most previous immunoassays. Although the impedance-based immunosensor showed a lower LOD (3.7 × 10−17) M,17 our biosensor exhibited a linear response for a wider range of MCLR concentration as compared with the former (1.0 × 10−16 − 8.0 × 10−15 mol/L). The capacitive immunosensor also achieved a lower LOD (7.0 pg/L) than our method, but required Ag nanoparticles for signal enhancement.25 It should be noted that the present method of detection does not require any labeling or modification of MCLR and antibody or signal enhancement factors. The present label-free detection scheme provided many advantages: (a) the whole assay time was reduced to 60 min, which would provide a much more rapid and convenient approach for the quantitative analysis of MCLR compared with traditional immunoassays; (b) the absence of complex labeling or modification steps for small antigen MCLR or antibody would allow a much better antibody−antigen interaction, thus realizing facile and sensitive detection of MCLR in water samples, which also improved the reproducibility and precision; (c) the antibody was attached onto the MCLR-immobilized SWCNTs by the antigen−antibody interaction, which enables the regeneration of the sensor simpler as compared with the direct immunoassay. The present LOD is 3 orders of magnitude lower than the standard (1 μg/L) set by WHO requirements for MCLR concentration in drinking water and

Figure 5. Responses of SWCNTs chemiresistive immunosensor for MCLR, MCRR, and MCLW. The concentrations of MCs were 10 ng/ L and incubation period was 1 h at room temperature. Each data point is an average of measurements from five independent sensors and the error bars represent ±1 standard deviation. R0 is the resistance of the SWCNTs channels after the immobilization of anti-MCLR antibody, R is the resistance of the immunosensors after incubation with MCLR, MCRR, or MCLW. E


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was directly bound to SWCNTs in the sensing channel, followed by a displacement assay. This method did not involve any labeling or modification of MCLR and antibody, thus simplifying the assay procedure and improving the antibody− antigen binding and achieving good sensitivity, selectivity, reproducibility and stability. Furthermore, the detection platform was successfully applied for the detection of MCLR in real samples. The proposed method provides a powerful platform for rapid and sensitive analysis of MCLR in environmental samples.

specificity for MCLR. Thus, the immunosensor provided the potential to selectively quantify the MCLR levels with high precision in environmental water samples. Reproducibility and Stability of Chemiresistive Immunosensor. The reproducibility of the label-free immunosensor was evaluated by using nine sensors for seven repeated binding cycles of the anti-MCLR antibody and 10 ng/L MCLR within 1 day. The coefficient of variation with one sensor ranged from 4.1% to 23.4%, and the average intraday coefficient of variation of nine sensors was 13.6%, showing good reproducibility. To evaluate the stability of the immunosensor, four repeated binding cycles of MCLR antibody and 10 ng/L were carried out within 1 week, that is, one measurement on days 1, 3, 5, and 7, respectively. When the immunosensor was not in use, it was stored at 4 °C. The biosensor showed no decrease in sensitivity and the coefficient of variation with one sensor ranged from 9.0% to 23.8%, with 14.2% of average intraday coefficient of variation of nine sensors. For the competitive binding mode, the immunosensor was regenerated by adding new antibody solution to compensate the antibody exhausted by sample MCLR. This method provides a moderate condition compared with previous immunoassay using acid or salts to dissociate the antibody−antigen complex.16 Based on the data of repeated binding cycles of antibody and MCLR as above, the present immunosensor showed excellent regeneration and thus good reusability. Analysis of Water Samples. To evaluate the suitability of the present method for practical applications, the immunosensor was applied to detect MCLR levels in water from tap, lake, and river with different background matrices. The water samples collected were simply centrifuged to remove any large suspended particles. The initial detection showed no presence of MCLR in the samples. In this case, the water samples were spiked with 10 ng/L and 100 ng/L (final concentration) MCLR standard and were subjected to the detection again. The analytical results are shown in Table 2. We observed that the

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Corresponding Authors

*(F.T.) Phone: +86-411-84707965; fax: +86-411-84707965; email: [email protected]. *(A.M.) Phone: +1- 951-827-6419; fax: +1- 951-827-5696; email: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the financial support of W. Ruel Johnson Chair in Environmental Engineering and NSF Water Sense (GRANT NUMBER: 1144635). We acknowledge Dr. Yingning Gao and Mr. Thien-Toan Tran for help in preparation of the sensor and Ms. Claudia Villarreal for help in SEM analysis.

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tap water tap water river water river water lake water lake water a

original conc.

spiked conc. (ng/L)

nd

10

nd

100

nd

detected (ng/L)

recoveries

RSD (n = 4−5)

96.5%

9.1%

112.6

112.6%

11.7%

10

10.2

102.2%

18.6%

nd

100

124.2

124.2%

18.7%

nd

10

84.7%

18.2%

nd

100

104.7%

15.8%

9.65

8.47 104.7

REFERENCES

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Table 2. Analytical Results of Spiked Real Water Samples by the Proposed Methoda samples

AUTHOR INFORMATION

nd: not found.

developed assay displayed good recoveries ranging from 84.7% to 124.2% for the real samples along with a relative standard deviation (RSD) ranging from 9.1% to 18.7%. These results indicate the potential applicability of the immunosensor for the quantification of MCLR in real waters. In conclusion, we have fabricated a SWCNTs-based labelfree chemiresistive immunosensor by displacement assay of the immobilized antibody on SWCNTs for sensitive and specific detection of MCLR in water. MCLR, being a small molecule, F


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