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Bisphenol-A sensors on polyimide fabricated by laser direct writing for on-site river water monitoring at attomolar concentration Cheng Cheng, Shutong Wang, Jayne Wu, Yongchao Yu, Ruozhou Li, Shigetoshi Eda, Jiangang Chen, Guoying Feng, Benjamin J. Lawrie, and Anming Hu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03743 • Publication Date (Web): 28 Jun 2016 Downloaded from http://pubs.acs.org on June 30, 2016

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ACS Applied Materials & Interfaces

Bisphenol-A sensors on polyimide fabricated by laser direct writing for on-site river water monitoring at attomolar concentration Cheng Cheng†, Shutong Wang, ‡⊥ Jayne Wu, †* Yongchao Yu‡, Ruozhou Li‡, Shigetoshi Eda,‡ Jiangang Chen,§ Guoying Feng,⊥ Benjamin Lawrie∥ and Anming Hu‡#* † Department of Electrical Engineering and Computer Science, The University of Tennessee, 1520 Middle Drive, Knoxville, TN, 37996, USA ‡ Department of Mechanical, Aerospace and Biomedical Engineering, The University of Tennessee, 1512 Middle Drive, Knoxville, TN, 37996, USA // Department of Forestry, Wildlife and Fisheries, The University of Tennessee Institute of Agriculture, 2431 Joe Johnson Drive, Knoxville, TN, 37996, USA §Department of Public Health, The University of Tennessee, 1914 Andy Holt Avenue, Knoxville, TN, 37996, USA ⊥ College of Electronics and Information Engineering, Sichuan University, No.24 South Section 1, Yihuan Road, Chengdu, 610065, PRC ∥ Computing Science and Engineering Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831, USA

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# Institute of Laser Engineering, Beijing University of Technology, 100 Pingleyuan, Chaoyang District, Beijing 100124, PRC Keywords: capacitive sensing, AC electroosmosis, aptasensor, point of care, laser direct writing

ABSTRACT: : This work presents an aptamer-based, highly sensitive and specific sensor for atto- to femto- molar level detection of bisphenol A (BPA). Due to its widespread use in numerous products, BPA enters surface water from effluent discharges during its manufacture, use, and from waste landfill sites throughout the world. On-site measurement of BPA concentrations in water is important for evaluating compliance with water quality standards or environmental risk levels of the harmful compound in the environment. The sensor in this work is porous, conducting, interdigitated electrodes that are formed by laser induced carbonization of flexible polyimide sheets. BPA-specific aptamer is immobilized on the electrodes as the probe, and its binding with BPA at the electrode surface is detected by capacitive sensing. The binding process is aided by AC electroosmotic effect that accelerates the transport of BPA molecules to the nanoporous graphene-like structured electrodes. The sensor achieved a limit of detection of 73.17 aM with a response time of 20 seconds. The sensor is further applied for recovery analysis of BPA spiked in surface water. This work provides an affordable platform for highly sensitive, real time, and field-deployable BPA surveillance critical to the evaluation of the ecological impact of BPA exposure.


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1. INTRODUCTION Recent years have seen an increasing interest in developing effective tools for the detection of small molecules in the environment and food supply. Bisphenol A (BPA) is a monomer widely used in a variety of consumer products, including plastics, food packaging, dental sealants, and thermal receipts. It was reported that 8-15 billion pounds of the material were produced annually.1 BPA has been detected in aquatic environments,2,3 river sediments, and fish muscles. Because BPA is a known endocrine disruptor with estrogenic, antiandrogen properties and can disrupt thyroid function, there is concern that the consumption of fish could contribute to the total intake of BPA in human populations. Detection and quantification of BPA concentrations in water are critical for the U.S. Environmental Protection Agency to develop water quality standards and to evaluate environmental risk levels for the protection of human health. The majority of current BPA detection methods rely on high performance liquid chromatography (HPLC) or enzyme-linked immunosorbent assay (ELISA), which require the use of costly instruments by skilled personnel with complex and time-consuming operation.4 Therefore, a simple and affordable method to monitor BPA level in environmental samples with high sensitivity remains critical. Many types of BPA biosensors have been reported, including bacterial biosensors utilizing engineered Escherichia coli with ligand-binding domains of estrogen receptor,5 electrochemical-cell-based chip sensors,6 surface plasmon resonance (SPR) sensors based on indirect competitive immunoassays,7 quartz-crystal-microbalance (QCM) sensors,8,9 carbonnanotube (CNT) sensors,10 and chitosan-based electrochemical sensors.11 However, these reported sensors were responded to doses in either µM (bacterial sensors and QCM sensors) or nM (electrochemical-cell-based sensors and CNT sensors) range, while BPA in practical samples often exist at pM level or even lower12,13. In order to reach lower limit of detection (LOD), aptamer probes, nanostructured sensors as well as labels have been increasingly used for the development of ultrasensitive assays. The small size of aptamers relative to antibodies 3 ACS Paragon Plus Environment


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is highly beneficial for ultra-trace capacitive sensing. Another advantage of aptamer probes is that they are much more stable and remain active under field-deployable conditions.14,15 BPA detection based on graphene oxide and anti-BPA aptamer and field-effect transistor (FET) sensor using aptamer modified multichannel carbon nanofibers (MCNFs) were also reported to detect BPA successfully in a pico or even femto level.16,17 Fluorescent labels were also be used in aptamer-based sensors for BPA detection. In the aptamer-based sensor reported by Ragavan et al,18 fluorescent and quencher molecules were functionalized on the aptamer, and changes in conformational change induced by BPA binding led to changes in the fluorescence emission proportional to the concentration of BPA. This method reached an LOD of 0.01 ng/L, or 43.8 fM, which was a significant improvement over a previously reported fluorescence immunosensor’s LOD of 14 ng/L, or 61.3 pM.19 Pico Molar detection limits have also been achieved with electrochemical aptasensors, however, they still require label particles20–22 as well as complicated experimental design and complex data interpretation processes.23–25 Table 1 summarizes the characteristics of various BPA sensors. Table 1. Performances of reported methods. Detection Method 5

LOD

Labeled

Response Time

Bacterial Biosensor

1.3uM

Yes

16-20 h

Electrochemical-cell-based sensor6

100 nM

No

24 h

7

100nM

No

1h

1 µM

No

2h

Immunoassay based SPR sensor

Quartz-crystal-microbalance sensor9 10

Carbon-nanotube (CNT) sensor

Chitosan-based electrochemical sensors11

74 nM

No

6h

10 nM

No

15 min

AuNP-based aptasensor14

438 pM

No

20-30 min

Aptamer-based electrochemical sensor15

1.24 pM

No

30 min

Field-effect transistor (FET) sensor

219 pM

Yes

15 min

Fluorescent molecules labeled18

43.8 fM

Yes

N/A

16,17

Fluorescence immunosensor

19

61.3 pM

Yes

15 min

ACEK Based capacitive sensing4

10 fM

No

1 min

This technique

73.17 aM

No

20 s

Recently, a label-free capacitive sensing method based on alternating current (AC) electrothermal effect was developed for BPA detection, which demonstrated good sensitivity, 4 ACS Paragon Plus Environment

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short response time and simple operation, highly suitable for on-site monitoring.4,26 In References 3 and 18, BPA was suspended in phosphate buffered saline solution and microfabricated electrodes were used as the sensor. To further reduce the sensor cost and improve the LOD for water monitoring, laser printed electrodes on polyimide were adopted as the sensor in this work and a different AC electrokinetics mechanism, AC electroosmotic (ACEO) effect, was used for more effective transport of the target analyte in water. In this work, ACEO -based capacitive sensing was used to detect the binding of BPA with aptamer at the electrode surface. ACEO is one type of AC electrokinetic (ACEK) effects, which are induced by applying an inhomogeneous field to microelectrodes in electrolytic fluids.27 ACEK has long been used for enrichment of analytes in biosensing application.28–32 However, in those biosensors enrichment and detection are separate33,34, therefore multiple steps are required, adding operation complexity. ACEO-based capacitive sensing incorporates analyte enrichment by ACEO effect with direct measurement of the fluid/electrode interfacial capacitance change in a single-step operation. ACEO-based capacitive sensing measures the interfacial capacitance continuously at a fixed AC frequency and voltage that is optimized for inducing ACEO effect and also allows for direct read-out of interfacial capacitance change without complicated data interpretation. ACEO effect can induce microfluidic vortices above electrodes to transport target molecules to the electrode surface for binding,35–38 which improves the detection sensitivity and response time.

ACEO flows are caused by the

movement of induced charges in the electrical double layer (EDL) at the solid-liquid interface when a non-uniform AC electric field is applied. The induced counter-ions will move under a tangential electric field to generate ACEO flows. ACEO velocity is known to exhibit a bellshaped dependence on frequency due to the charging process of EDL.39 Therefore, the frequency of capacitive sensing needs to be optimized to maximize ACEO effect.



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To increase sensor sensitivity, introducing a porous nanostructured surface layer is one of the most effective methods that increases the absorbed volume of target molecules. Interdigitated sensors fabricated by conventional manufacturing methods, such as thin film deposition, screen printing or electroplating combined with etching40,41 have limited surface areas due to micro-sized grains in electrodes. Electrode-patterning by printing including inkjet printing is one attractive option for the fabrication of sensors due to their relative high-speed, facile operation, ability to produce 3D structures, and compatibility with various substrates.41– 43

However, in inkjet printing, the material selection of ink is limited to Ag, Cu, Au and

conducting carbon, and the ink fabrication is also complicated.44,45 There are many reports to increase sensitivity to modify electrode surface with various nanostructures, such as conducting polymer, metal or oxide nanoparticles, Carbon nanotube (CNT)46 or graphene.40 Unfortunately, tedious surface engineering has to be done with extra processing. Recently, laser direct writing (LDW) of polyimide sheets with CO2 lasers or femtosecond lasers has been demonstrated in the manufacturing and processing of flexible polymer devices.47–50 Conductive circuits and porous structures can be simultaneously realized in simple one-step fabrication. In order to further advance this technique to achieve ultrasensitive biosensing, in this work we successfully integrated one-step LDW to fabricate flexible and highly sensitive interfacial capacitance sensors with porous carbon structures and demonstrate their versatile use in atto- to femto- molar level detection of BPA.

2. EXPERIMENTAL SECTION 2.1. Sample Preparations. . Washing buffer 0.1x PBS is prepared by 1:10 volume dilution of physiological strength PBS in ultrapure water to obtain 1 mM phosphate buffer (pH 7.0) containing 15 mM sodium chloride. Blocking solution is 1.0 mM 6-mercaptohexanol in ultrapure water. 20 µM aptamer in ultrapure water is used for incubation. Analytical BPA


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samples are prepared as 0.1 fM, 1.0 fM, 10 fM, 100 fM and 1 pM BPA in ultrapure water. Surface water samples were collected from Tennessee River near a waste water treatment plant in Knoxville and were diluted at 1:1,000,000 in ultrapure water prior to testing. No filtering was performed. 2.2. Laser Writing Electrode Sensors. The laser written electrodes used in this work were fabricated by a femtosecond (fs) pulsed laser (Cazadero, Calmar Laser Inc.) and a 532 nm CW laser (Verdi G5, Coherent) via a 20X microscope objective (NA=0.42) on the flexible Polyimide (PI) sheet (Kapton_ HN, 100 µm thickness). The fs laser generates pulses with 1030 nm central wavelength, and 400 fs pulse duration at a repetition rate of 120 kHz. The PI films are mounted on an XY-translation stage controlled by a computer. In our case, the fs laser and CW laser power concentrated on the sample surface are 230 mW and 85 mW with a spot size of 3-5 µm, respectively, and a scanning speed of 0.5 mm/s. All irradiation was performed in air environment under normal incidence. The gap distance between adjacent finger electrodes was fixed at 90 µm, and a total of 8 pairs of electrode fingers were written in one sensor. The generation of black lines on the polyimide sheet after the laser scanning confirmed the carbonization of the polymer. Prior to irradiation, the PI film was cleaned using ethanol. Finally, the surface morphology was observed using optical microscopy and scanning electronic microscopy (Zeiss Auriga). After surface treatment by plasma cleaner or ozone cleaner, a microchamber is attached to the foil surface for surface functionalization and subsequent sensing. 2.3. Electrodes Surface Functionalization. The electrode’s functionalization after plasma treatment included receptor incubation and uncovered surface blocking. As the incubation and blocking processes progressed, an increasing number of molecules became attached to the electrode surface. The quality of electrode functionalization was monitored by measuring the Cint. This method was validated in our prior work.51 During the incubation and



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blocking process, Cint values have reduced by -58.05±3.19% and -93.35±2.06% respectively, indicated adequate molecular immobilization on the electrode surface. Surface immobilization reduced the current flowing through the electrodes, effectively increasing the charge transfer resistance, Rct. Therefore Rct was also measured during the incubation and blocking, which showed an increasing Rct with time. The data plot can be found in Figure S4 in the supplementary material. 2.4. Measurements and Data Analysis. This work uses ACEO-based capacitive sensing to monitor the molecular deposition at the electrolyte/electrode interface. Prior work demonstrated that interfacial capacitance (Cint) can effectively detect molecular deposition on the electrode surface with high sensitivity and specificity52–54. In ACEK capacitive sensing, Cint is found by measuring the sensor cell’s impedance at a fixed and non-biased AC frequency and voltage continuously during the testing. The interfacial capacitance of the electrodes was sampled and recorded periodically by an Agilent 4294A impedance analyzer for 20 seconds. Then the percentage change, dC/dt in %/min, of the measured capacitance was adopted as the readout of the sensor, indicating the binding reaction occurring on the electrodes surface. Least square linear fitting algorithm was performed to determine the capacitance change rate.

3. RESULTS AND DISCUSSION 3.1 Microstructure and surface carbonization. The sensors were fabricated by using a femtosecond laser and a continuous wave (CW) laser to irradiate a commercial flexible polyimide (PI) sheet under ambient conditions, as shown in Figure 1 (a). The two beams were overlapped and tightly focused onto the sample surface in order to integrate the multiphoton effect from the femtosecond source and the thermal effect from the CW source.55 The geometrical parameters for laser scanning are showed in Figure 1S in supporting information. Although the size of focal point is around 3 micrometer based on calculation, the real size of 8 ACS Paragon Plus Environment

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irradiated regions is about 80 micrometers due to the significant thermal effect. The black line in Figure 1 (b) illustrates the flexible electrode patterned by the laser, and the light orange regime indicates pristine PI. Interfacial capacitance sensors were patterned with the combination of a 230 mW femtosecond (fs) pulsed fiber laser at a wavelength of 1030 nm and 85 mW green laser beam at 532 nm. The sensors were further characterized with SEM and Raman spectroscopy. Meanwhile, the sheet resistance of the electrode reduced to

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