Microsystem Lab-on-a-Chip device for detection of antibiotics in milk

Lab-on-a-Chip takehome exam 1, by Caralee Nelson, Xuan Li and Morten Hilligse 22. March 2013 All authors contributed equally.

Table of Contents
Introduction ....................................................................................................................................................... 3 Antibiotics1,2................................................................................................................................................... 3 Motivation2 .................................................................................................................................................... 3 Targeted audience ......................................................................................................................................... 3 Specifications ................................................................................................................................................. 3 Techniques......................................................................................................................................................... 5 Electrochemical Impedance Spectroscopy .................................................................................................... 5 Aptamers ....................................................................................................................................................... 6 Design ................................................................................................................................................................ 7 Microsystem .................................................................................................................................................. 7 Electronic reader ........................................................................................................................................... 8 Fabrication ......................................................................................................................................................... 9 Quality evaluation ........................................................................................................................................... 12 Testing time ................................................................................................................................................. 12 Sensitivity..................................................................................................................................................... 12 Selectivity .................................................................................................................................................... 13 Signal to noise (s/n) ratio............................................................................................................................. 13 Reliability ..................................................................................................................................................... 14 References ....................................................................................................................................................... 14

Introduction
Antibiotics1,2
Antibiotics are substances that kill or inhibit growth of bacteria. Since the 1940s, antibiotics have been used to treat diseases in humans, including conditions such as ear and skin infections and food poisoning, and antibiotics save millions of lives each year. Antibiotics belong to a drug category called antimicrobials, which is further divided into classes which describe the origin of the antibiotic drug, such as penicillins, tetracyclines and ionophores. Antibiotics are often small molecules, and many of todays antibiotics are semisynthetic modifications of natural compounds, isolated from living organisms.

Motivation2
Unfortunately, various bacteria strands responsible for a number of human diseases are becoming increasingly drug-resistant. This means that infections require new antibiotics for treatment, or in the worst case, are untreatable. Some of this bacterial resistivity is thought to come from the use of antibiotics in the modern food animal industry. In the food animal industry, antibiotics are not only used to treat diseases, but are also used as growth-promoting food additives. Between 1985 and 2001, the use of antibiotics in livestock feed rose by 50%,3 and today, antibiotics are routinely fed to livestock, poultry, and fish to promote faster growth and to compensate for unsanitary conditions in which they may be raised4. Although the use of antibacterial food additives has been banned in the EU since 2006, it is still widely used in the rest of the world.5, 6 The use of low doses of antibiotics allows drug-resistant bacteria to emerge on farms, which can then reach the population in various ways. EU has regulations on the amount of antibacterial concentration in food products. 7 We have tried to develop an electrochemical Lab-on-a-Chip device that can measure the concentration of various antibiotics in milk, which will make it easy to test whether the regulations are met.

Targeted audience
The targeted audience of our Lab-on-a-Chip device are the farmers and the local dairy. The device should allow the farmer to directly measure the concentration of a given antibiotic compound in the freshly tapped milk, and allow any dairy worker to measure on the delivered milk, without the need of expensive equipment and advanced measurements.

Specifications
The chosen target group sets a number of requirements on the device. First of all, the device should be so easy to use that no training is required to operate it. Secondly it should be cheap enough that every dairy farmer can afford it, regardless of whether he owns 20 or 200 cows. Other requirements are sensitivity, specificity and reliability, which also need to be met. The sensitivity should be high enough that concentrations well below that regulation limits can be measured. Because multiple antibiotics are often used on farms, the specificity needs to be high enough that only a single antibiotic substrate is measured. And of course the device needs to be reliable enough that separate measurements will yield the same result.

Because of these specifications, it was chosen to make a device based on impedance spectroscopy, with aptamers as immobilization agents. This allows for label-free measurements of antibiotic substrates, which mean that no sample preparation is required, and no chemicals need to be handled by the end user. The technique of impedance spectroscopy and aptamers are described in the next section. Because the antibiotics of a sample will bind to the aptamer-functionalized electrodes, this method is most suitable for single use devices, where the system is discarded after use. Therefore, an effort has been made to design a system and a fabrication method that results in low production cost. Modern glucose meters have been used as inspiration for this. In a glucose meter, blood is sucked into a channel with electrodes on a small plastic strip, which is then placed in an electronic reader device. The electronic reader will then measure glucose level of the blood sample, and when the measurement is done, the strip is discarded. This allows for a very small and simple system, which is cheap enough to be discarded after a single use. Figure 1 shows a modern glucose meter.

Figure 1 Modern glucose meter with test strip inserted at the bottom.

Techniques
For this lab on a chip system, electrochemical impedance spectroscopy (EIS) is used with electrodes functionalized with oligonucleotide aptamers that shows high affinity of 3 common antibiotics in milk.

Electrochemical Impedance Spectroscopy

We use the electrochemical detection method of EIS, because it is label-free, highly sensitive, and fast. EIS is a powerful method for analyzing the complex electrical resistance of a system and it is sensitive to surface phenomena and changes of bulk properties. The impedance of a circuit supplied with an alternating current, is defined as the voltage divided by the current. The impedance is usually represented as an imaginary number, defining the phase shift of the potential and current. The general technique of EIS is to apply a low voltage alternating current with changing frequencies, and measure the impedance response of the system. Often, there is an equivalent circuit model, composed of resistors and capacitors, fitting the electrical properties of the sensor. The system impedance is highly sensitive to the surface reactions, bulk properties, disturbance of the double layer (the double layer can be seen as a capacitor, and its capacity mainly depends on adsorption of particles on the surface and concentration of the ions), and geometrical capacitance of the electrochemical system. This is illustrated by Figure 2. By functionalizing the electrodes with aptamer that shows high affinity toward an antibiotic, the antibiotic will bind to the aptamers, thereby causing surface change of the electrode and bulk properties.

Figure 2 A representative circuit for electrodes in solution. Solutes close to the electrodes will affect the capacitances.8

The electrodes of this Lab-on-a-Chip system will be connected to a portable reader device, which consists of a viewing screen, a voltage frequency generator and an ammeter. The reader device will then measure the impedance of the electrodes over a range of frequencies. A frequency range of 100kHz to 200mHz, on a logarithmic scale with 10 points per decade for frequencies 66 Hz and four points per decade for frequencies 66 Hz, has previously been used to measure the affinity binding of antibiotics to aptamers by Johannes Dapr et al 2013.9 By recording a baseline with no antibiotics present, and then recording spectras of increasing concentrations, the change of the impedance signal will be recorded and thus forming a working curve. In practical application, the concentration of different antibiotics in the milk sample will be derived from the working curve according to their impedance signal.

Aptamers
Here we use oligonucleotide aptamers, which can specifically bind to different antibiotics with high affinity as the binding sensors. The aptamers are single strand, short sequence DNA or RNA, and they can typically fold into a three-dimensional structure, whose conformation can change upon binding. We choose to use oligonucleotide aptamers because their sequences have already been developed, and they are commercial available. In addition, there are plenty of successful methods to functionalize the electrodes with aptamers by simple steps. Whats more, compared to antibodies, oligo-peptides, or enzymes, aptamers are more stable, not so sensitive to pH or other environmental changes, and thus can keep a longer activity. The high affinity aptamers for the three target antibiotics have been successfully developed by the SELEX (systematic evolution of ligands by exponential enrichment), which enables the selection of high-affinity nucleic acid sequences from a random pool of candidates. The aptamer sequences of this lap-chip system are shown below: Ampicillin (Kyung-Mi Song et.al 201212) 5-GCGGGCGGTTGTATAGCGG-3 Tetracyclines (Javed H. Niazi et al. 200813) 5-GGGCGGGGGTGCTGGGGGAATGGAGTGCTGCGTGCTGCGG-3 Streptomycin (S T Wallace and R Schroeder et.al 199814) 5-GGAUCGCAUUUGGACUUCUGCCCAGGGUGGCACCGUCGGAUCC-3 All the aptamers are synthesized by BGI (Shenzhen Huada Genomics) as high performance liquid chromatography (HPLC) purified and lyophilized ssDNA or ssRNA functionalized with a 5amino modified C6 linker. Apart from using sequences according to the articles, the aptamers for the three antibiotics above have already been commercially available. The company Aptagen sells aptamers for different antibiotics15.

Design
Recently, antibiotics in milk have been measured using impedance spectroscopy, by immobilizing antibiotic binding aptamers on the electrodes9, 10. The method has been demonstrated on both gold electrodes and polymer electrodes. By using these methods and combining them with inspiration from modern glucose meters, we have designed a user-friendly Lab-on-a-Chip system. The system is shown in Figure 3.

Microsystem
The system consists of a single channel with dimensions of 8.2mm in length, and 200m in width and height, which sucks up the sample through capillary action. The channel is open on both ends to allow air to escape. Inside the channel is a series of electrodes according to the design of Dapr et al.,9 with 5 cathode pins and 5 anode pins each of 20m in width and 300m in length, where 200m of each pin is exposed to the sample. Each electrode pair is then functionalized with a different immobilized aptamer which will bind a specific antibiotic. Aptamers binding the antibiotics of ampicillin, tetracycline and streptomycin were chosen because they are commonly used antibiotics, and aptamers are available that will bind to these substrates. The system is made up of a bottom and a top part, with the channel being defined in the top part, and the electrodes applied to the bottom part. The top part is smaller in length than the bottom part, which exposes the electrodes.

Figure 3 Semi-transparent view of the designed microsystem.

Figure 4 Total overview of the designed system.

Figure 5 The electrode pattern used, with electrode widths of 20m.

The idea is that the opening of the channel (top left end in figure 3) is dipped in the sample, which will suck up milk through capillary action. After this, the exposed electrodes of the system is easily connected to an electronic reader device by insertion of the system, in the same way as known from glucose test strips. It should be noted that the design imaged in figures 3, 4 and 5 has total dimensions of 13.6mm x 6.0mm x 0.45mm, which might actually be too small for people to handle, so extra plastic should be added for proper handling of the microsystem.

Electronic reader
After the microsystem has been loaded with the milk sample via exposure of the channel end, the other end of the device, which contains the exposed electrodes, is plugged into the electronic reader the same way as one would insert a USB device in a USB port. This reader would have a digital screen that would display the concentration and name of each antibiotic. A database of the legal limits of common antibiotics could also be included on this reader. Once the measurement data has been recorded by the user, the microsystem can be pulled out from the reader and disposed of.

Fabrication
The microfluidic system is made from some of the same polymer components as that of Dapr et al. (2012):9 the base and lid are made of Cyclic Olefin Copolymer Topas, and the electrodes are composed of the conducting polymer PEDOT-OH:TsO. Polymers were chosen because they are well-suited for disposable devices: such polymer devices are inexpensive, reliable, and can perform rapid analysis.9 A conductive polymer was chosen for the electrode material as it is cheaper than the conventional noble metal electrode, inexpensive to fabricate, easy to functionalize, and highly biocompatible. The fabrication of the microsystem begins with a Topas wafer base. A conductive film of EDOTOH:TsO is deposited by spin coating Baytron Ca, butanol, pyridine, and EDOT-OHb in the same method described in Dapr et al. (2012). The monomer EDOT-OH:TsO is then polymerized to PEDOT-OH:TsO via thermal activation during nanoimprint lithography (NIL), using the heated NIL mold to polymerize the monomer upon pressing (fig 7). This design differs from Dapr et al. (2012) in that it uses only a single layer of PEDOT-OH:TsO rather than a bilayer composite of PEDOT:TsO and PEDOT-OH:TsO. The PEDOT-OH:TsO seems to be the functional layer that provides the hydroxymethyl groups used in electrode functionalization, so this polymer was chosen. The reason for the bilayer is not elaborated upon in Dapr et al. (2012) but we theorized it could be related to greater binding stability; that is, perhaps the non-hydroxymethylated PEDOT binds better to Topas and PEDOT-OH binds well to PEDOT. However, being unsure of the true purpose the bilayer, we chose to simplify the process and use only the hydroxymethylated polymer. The NIL technique was chosen for fabrication of the microelectrodes because it allows for the imprinting of a very small and shallow structure into the conductive layer but is not too expensive or time-consuming. Particularly, the ability to create shallow protuberances is important because we do not want our electrodes to be too tall. Tall electrodes could interfere with the placing of the electrode-functionalization well frame, giving a gap that could cause leakage upon addition of the functionalization solution (although we have taken measures against this by choosing a soft, conforming, hydrophobic frame material; as described in more detail later). In this case, the EDOT-OH:TsO serves as the thermoplast layer, which is imprinted with a mold (figure 7a,b). The heated mold performs the duel duty of polymerizing the thermoplast into PEDOT-OH:TsO and shaping it into the electrode structure. In the areas imprinted by the mold, the thermoplast is pushed away, creating indentations. The thermoplast is pushed into the areas not imprinted by the mold, turning these areas into protuberances.11 Any leftover thermoplast remaining in the valleys (typically only a few monolayers thick, figure 7c) is removed with the dry etching technique, plasma etching (figure 7d). Plasma etching was chosen as it is safe and appropriate for the small amount of thermoplast that must be removed.

a b

40% Fe(III) tosylate in butanol. ((2,3-dihydrothieno[3,4-b][1,4]-dioxin-2-yl)methanol) or hydroxymethyl-EDOT.

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Plasma etching works based on the dissociation of gas molecules into electrons and ionized radicals, under conditions of low pressure, low temperature, and radio frequency power supplies. Material can be removed via chemical etching, physical etching, or physiochemical etching (whereupon both chemical and physical etching are applied). Chemical etching usually creates isotropic walls and physical etching produces anisotropic walls. To create vertical sidewalls, one can use physiochemical etching with sidewall passivation. Here, a fluorocarbon is used as the etching gas. The main problem with using this sidewall passivation technique is that fluorocarbons such as CHF3 are greenhouse gases. We chose to use O2 gas in the plasma ashing technique; however, this produces isotropic profiles, but because we only need to remove a few monolayers, it shouldnt be a problem.

Figure 7 Nanoimprinting lithography technique.

A well frame made of poly(dimethylsiloxane) (PDMS) containing three microwells is then aligned onto the electrodes, so that each well exactly matches an electrode, as done in Xu et al. (2005)10 (figures 8 and 9). PDMS is a soft, conforming, hydrophobic material that fits over the electrodes to form a watertight seal; thereby preventing the height of the electrodes from creating a gap that would cause leakage of the functionalization solution.

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Figure 9 Frame used for electrode functionalization.

Figure 10 Semi-transparent view of figure 9.

Once the well frame is in place, the electrodes are functionalized as in Dapr et al. (2012). Succinic acid is grafted onto the surface hydroxymethyl groups of the PEDOT-OH:TsO using the coupling agent EDCc, and the resulting carboxylic acid groups are activated with EDC. Then in each microwell, the corresponding 5-amino modified aptamer is added to form a stable amide bond and left to react for three hours9. After immobilization, the frame is removed, and the aptamer-modified chip is rinsed and dried. The top segment of the microsystem, the Topas lid, is created using the hot embossing technique. Topas is heated to the glass transition temperature, pressed with a mold to form the microchannels, and then cooled down below the glass transition temperature again. This is a cheap, fast technique that can faithfully reproduce microstructures at the nanoscale level.11 Finally, the top and bottom segments of the microsystem are sealed together using thermal bonding. The pieces are pressed together at an elevated pressure and temperature as in Dapr et al. (2012).

1-ethyl-3-(3-dimethylaminopropyl)carbodiimide.
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Quality evaluation
Testing time
The testing time of our chip is determined by the speed of the binding reaction, diffusion coefficient of the antibiotics, diffusion distance decided by the channel dimension, and the impedance reading speed of the impedance spectroscopy device. To figure out the testing time, we do not need to know the number of each parameter and combine them mathematically. By doing impedance spectra at several times, we can analyze the impedance data curve to figure out how much time it takes for the impedance curve to stabilize, which gives us the testing time. This is done by Dapr et al. as shown in figure 11. The measurements show that changing the concentration of antibiotic, will affect the impedance, which in turn will stabilize within approximately 20min. This shows that the analysis time is reasonably short for a number of real-life applications.

Figure 11 Time dependent Impedance measurements

Sensitivity
The maximum residue limit (MRL) for the three target antibiotics according to EU regulation no. 675/92 (as amendment to regulation no. 2377/90) are shown in the table below. Antibiotic EU Maximum Residue Limit in Milk (g/kg) 4 100 200 EU Maximum Residue Limit in Milk (nM) 11.45 220.32 383.89

Ampicillin Tetracyclines Streptomycin

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As presented by Johannes Dapr et al.2013, EIS test using electrodes decorated by the same aptamer for ampicillin is able to detect the first statistically significant (p<0.05) signal at100pM, much lower than 11.45 nM, and further increase of concentration showed a fairly linear dependence in the logarithmic plot. Thus our lab-chip system is capable of reproducibly detecting an ampicillin concentration in milk that is far below the MRL set by the EU. As presented by Juankun Zhang et al.16, the minimum detectable amount of tetracycline is approximately 2nM by EIS test using electrodes decorated by a similar aptamer to what we use in our system. In the logarithmic plot, a linear range between 2.079 and 62.37nM is shown, and an identifiable curve between 2nM to 230nM is shown. Thus our lab-chip system can also be capable of reproducibly detecting a tetracycline concentration in milk that is far below the MRL set by the EU. But of course, a similar analyzing process needs to be carried out for our system, since the aptamer we use is not exactly the same as in the article. Up until now, there are no published articles demonstrating the detection of streptomycin by aptamer functionalized electrodes using EIS. But since the aptamers can bind the antibiotics with high affinity, the impedance of the system is highly sensitive to the binding reaction and surface change, and the maxium residue limit of streptomycin is even larger, we believe that our method is able to detect the streptomycin below the MRL set by the EU. Also, a similar testing process to analyze the sensitivity of the lab-on-chip system to streptomycin is recommended.

Selectivity
One of the good properties of oligonucleotide aptamers is that they can bind to different target antibiotics with high affinity and specificity. And the sequences of the three aptamers are successfully developed by the SELEX (systematic evolution of ligands by exponential enrichment), which enables the selection of high-affinity nucleotide acid sequences from a random pool of candidates. All of these guarantee that the oligonucleotide aptamers will perform with high selectivity for our antibiotics test. And the selectivity of the three aptamers has already been demonstrated by published articles.12, 13, 14

Signal to noise (s/n) ratio

In an aptamer-based electrochemical sensor, detection of the analyte is based on the signal generated by the conformational change of the aptamers17, so one of the factors controlling the signal to noise ratio is how the aptamers are folding. For one thing, if the conformation of the bound aptamer is sufficiently different than the conformation of the unbound aptamer, a strong signal will be generated. That is, a large difference in the conformation of the bound and unbound aptamer will make a larger signal. In addition, whether the aptamers are performing correctly will make a difference: correct folding of the aptamer will allow for high-affinity binding to the analyte, while misfolding and non-folding will give a low signal to noise ratio. Factors that affect aptamer conformation include incubation time and buffer ion composition17. As we used the same incubation time and buffer as in Dapr et al., and some of the same aptamers, we can presume that our signal to noise ratio will be similar.

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Reliability
The variance in accuracy and precision among microsystems will primarily depend on how welldefined the electrodes are. Although a relatively simple process, nanoimprint lithography does give a high resolution and can produce structures on a nanometer scale. There are some risks of defects: for example, bubbles can form when air is trapped between the mold and the thermoplast.18 However, in our electrode layout, there are no isolated areas of contact between an indentation in the mold and the thermoplast layer, so air will be able to escape upon stamping. Another source of defect is adhesion between the mold and the thermoplast: if some of the thermoplast stays on the mold after stamping, it will degrade the pattern and cause defects in the following chips. Adhesion can be prevented by coating the mold with a layer of FDTS, a desticking agent.19 With proper defect prevention measures, our fabrication method can be assumed to give reliability reproducible microsystems.

References
1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. http://en.wikipedia.org/wiki/Antibacterial (20-03-2013) http://www.gracelinks.org/257/antibiotics (20-03-2013) Gerber, P., Opio, C., & Steinfeld, H. Poultry production and the environment a review. Food and Agriculture Organization of the United Nations Emanuele, P., Antibiotic resistance. American Association of Occupational Health Nurses Journal, 58 (9) 2010 http://www.globalization101.org/regulating-antibiotics-in-animals/ (22-03-2013) http://www.allaboutfeed.net/Nutrition/Feed-Additives/2012/4/Russia-rapidly-increasing-use-ofantibiotics-in-feed-production-AAF013059W/ (20-03-2013) http://ec.europa.eu/health/files/eudralex/vol-5/reg_2010_37/reg_2010_37_en.pdf (22-03-2013) Class presentation by Noemi Rozlosnik Johannes Dapr, Lasse Holm Lauridsen, Alex Toftgaard Nielsen, Noemi Rozlosnik, Biosensors and Bioelectronics, 2013, 43, 315320 Danke Xu, Dawei Xu, Xiaobo Yu, Zhihong Liu, Wei He, and Zhenqiu Ma, Anal. Chem. 2005, 77, 5107-5113 Geschke, Oliver, Henning Klank, and Pieter Telleman, eds. Microsystem Engineering of Lab-on-aChip Devices. 2nd ed. Weinheim: Wiley-VCH, 2008. Kyung-Mi Song, Euiyoung Jeong, Weejeong Jeon, Minseon Cho, Changill Ban, 2012 Javed H. Niazi, Su Jin Lee, Man Bock Gu, 2008 Scott T. Wallace and Renee Schroeder, 1998 http://www.aptagen.com Juankun Zhang et al 2013 Wei Fang, and Chih-Ming Ho. "Aptamer-based Electrochemical Biosensor for Botulinum Neurotoxin." Anal Bioanal Chem 393 (2009): 1943-948. Liang, Xiaogan, Hua Tan, Zengli Fu, and Stephen Y. Chou. "Air Bubble Formation and Dissolution in Dispensing Nanoimprint Lithography." Nanotechnology 18.2 (2007): 025303 http://en.wikipedia.org/wiki/FDTS (21-03-2013)

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