Development and preliminary evaluation of novel materials for selective detection of oseltamivir in waste water

Development and preliminary

evaluation of novel materials for

selective detection of oseltamivir in

waste water

in waste water

1

Abstract

This project aims to investigate whether it is possible to detect consentrations down to 0,3 µg/L of oseltamirir in waste water by Quartz Crystal Microbalance. The risks linked to this contaminant could be not only environmental, scientists have found that infuence recistence can propagate due to contaminats of this kind. Cases similar to the bird- and swine-flu could occure if the drug spreads through animals and mutate.

Four systems of different character was synthesised. As a result it was found that MAA functional monomer systems probably was interfering with charges within it’s own nanowires.

Reference systems could not detect 0,01 mg/mL and HEMA polymer surfaces was the only reliable system, pH of 5,1. Lowest limit of detection (LOD) was found in Molecular Imprinted Polymer nanowires (MIP Nw) at 0,01 mg/mL and could be scaled down to 0,1 mg/L with increased loop size (injection at 10x that of 0,01 mg/mL at about 500 µL and a halved flow rate.)

An alternative could be nano structuring such as ”polystyrene balls” that possibly could be able to achieve surfaces with even more binding sites needed to detect the lowest limit at 0,3 µg/L.

Definitions

Novel materials e.g. polymers.

Thanks

2

Content

1 Introduction ... 3

1.1 Background ... 3

1.2 Objectives ... 4

1.3 Materials and Methods ... 5

1.3.1 The extraction of Oseltamivir ... 5

1.3.2 The making of MIP´s ... 5

1.3.3 The making of references ... 6

1.3.4 The method of FT-IR and preparing QCM analyse ... 7

1.3.5 QCM analyse method ... 8

1.4 Demarcation ... 8

2. Analyse results ... 10

2.1 Extraction and H-NMR Analyse ... 10

2.1.1 FT-IR analyse ... 10

2.1.2 QCM analyse ... 11

2.2 QCM Analyse results ... 11

2.2.1 HEMA QCM analyse results ... 11

3. Discussion ... 18

4. Conclusions ... 19

3

1 Introduction

The potential for spread dispersion of new strains of influenza with human pathogenicity or with impact on agriculture is being facilitated by anthropological activity, in particular the over-prescription of anti-viral agents and their release into water systems via the sewage systems [1]. Of particular interest in this respect are the small molecule weapons targeting the influenza virus capsid protein neuraminidase, namely oseltamivir (Tamiflu®)[2-4] and zanamivir (Relenza®) [5], which currently form our last line of defence against this virus. Clear evidence of the presence of these substances in the world’s water systems, a factor that can contribute to resistance development, has been a serious concern [6]. Recently, the identification of strains resistant to this class of substances has highlighted the importance of methods for the serious concern [7-9]. Accordingly, the development of methods for the rapid and sensitive determination of these substances in the environment is required. Real World monitoring situations necessitate that sensors are robust and are able to withstand the rigors (e.g. extremes of temperature, exposure to complex matrices) of environments not normally conducive to biomacromolecular stability. Ultimately, such materials can even be utilised for the selective absorption of these compounds from waste water.

The molecularly imprinting technique provides access to synthetic polymers with predetermined recognition sites through the synthesis of a polymer with suitable functionalities in the presence of a template, often the target or an analogue thereof [10]. These materials have recognition properties reminiscent to those of antibodies [11], though are significantly more stable than biomolecular structures, generally having high tolerances to extremes of temperature, pH, and organic solvents and have long shelf lives [12], thus making them excellent candidates for use as alternatives to their biological counterparts, e.g. in sensing or separation applications.

4 It was envisaged that combining these nano-structuring strategies with a recently described imprinted polymer system with selectivity for oseltamivir [17] could provide access to novel, robust and highly selective recognition materials for use in a surface-based sensing for monitoring the presence of oseltamivir in waste waters, e.g. using quartz crystal microbalance technology [14, 18]. In addition to providing a means for the sensitive and robust monitoring of oseltamivir in water ways, such materials may also, ultimately, offer promise for use in the purification of oseltamivir contaminated water as selective filters.

Maximum limit amount of oseltamivir was found in sewage water in Japan and was about 0,3 µg/L using HPLC, High pressure liquid chromatography [19].

1.2 Objectives

The primary objective of this study is to develop materials for the selective and sensitive monitoring of environmentally problematic substances in waste water and in natural water systems. Within the framework of this study, a series of novel synthetic polymers with oseltamivir-selective recognition properties shall be developed as sensor recognition elements and assessed for use in the determination of oseltamivir in waste water. In particular:

1. Molecularly imprinted polymers with selectivity for oseltamivir shall be prepared in thin film formats on quartz crystal resonators;

2. Resonators shall be used as quartz crystal microbalance recognition elements and limits of detection determined using oseltamivir-spiked water from local sites;

3. Time permitting, samples from Kalmar Vatten’s sewage works (Kalmar County Hospital) shall be analyzed with respect to oseltamivir content.

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1.3 Materials and Methods

1.3.1 The extraction of Oseltamivir

Firstly, extraction of oseltamivir shall take place as described in [17]. Some differences as the number of tablets - nine instead of thirty - and the quantity of repeated extractions will occur. Due to the split of tablets by one third, all substances will be split correspondingly. Additionally, only one filtration of the supernatant will be made after centrifugation. This will be made by a 0,45 µm filter since further accuracy is too fine-masked. Furthermore, proton nuclear magnetic resonance known as H-NMR will be used as method to show the grade of pureness in the extracted sample by interpretation of the diagram (Figure 2).

Figure 1. The structure of oseltamivir and how the ester is hydrolysed in the body as made in the

extraction to clean out the active molecule without ethanol. Extracted into organic form in chloroform solvent.

1.3.2 The making of MIP´s

Polymeric materials selective for oseltamivir shall be prepared by the molecular imprinting of oseltamivir in a series of cross-linked acrylate-based polymers based upon the previously reported protocols by [17]. The polymerization reactions shall be adapted for use in two thin film fabrication formats, both using Au-coated quartz crystal resonators as substrates.

6 The first following the general procedure established by [14] for ultra (≈5 nm) thin film preparation and the second for nanowire synthesis as described by [15, 16].

This shall be performed using 2,0 mg oseltamivir as template solved in 148 µL of the solvent porogen ACN, acetonitrile. This shall be vortexed and sonicated then divided into two falcon tubes containing 74 µL each. Two functional monomers, methacrylic acid (MAA) and 2-hydroxy ethyl methacrylate (HEMA) will be added; 4,1 µL MAA and 5,8 µL HEMA, one to each falcon tube. Furthermore, the cross-linker ethylene glycol dimethacrylate (EGDMA) will be added in 41,4 µL to the both samples and they both shall be vortexed. Lastly the initiator, 2,2´-azobis(isobutyronitrile) AIBN, will be added with a small spatula, approximately 2,5 mg to each tube. One drop (2 µL) of the mixture shall directly be applied, e.g. dropped onto two silicon wafer surfaces each. One each of which has got an anopore membrane layer.

The method will be used to make the mix and to apply it onto QCM-ships. The monomers will be added separately, MAA to one tube mix and HEMA to the other one.

The two silicon wafer surfaces with anopore membrane layers will be put into one beaker each containing 10 mL 1,2 M HCL and put into a 35 ºC water bath for more than 12 hours. The other two silicon wafer surfaces will be stored in one tube each. NaOH will be prepared in a concentration of 0,01 M for the molecule imprinted polymers (MIP’s) to bath in for 2 hours while whipped on a tilting-table. In between water bath and NaOH cleaning the MIP-surfaces shall be cleaned with water and air dried. The MAA and HEMA MIP Films (without anopore membrane) will only be cleaned in NAOH and Methanol. See table 2.

The four wafers shall be put under UV-light for about 3 hours surrounded by carton carboard walls to polymerize.

1.3.3 The making of references

7 Table 1. All four systems contain ACN, FM, EDGMA and EDGMA except what is shown in this table.

Table 2. Cleaning Method, (RT = room temperature)

REF FILM REF Nw MIP FILM MIP Nw

2hrs RT 0,01M NaOH 12hrs 35ºC 1,2M HCl 2hrs RT Methanol 12hrs 35ºC 1,2M HCl

2hrs RT Methanol 2hrs RT 0,01M NaOH 2hrs RT Methanol

2hrs RT Methanol

Lastly all wafers shall be put under UV-light for about 3 hours surrounded by carton carboard walls to polymerize nanowire surfaces and film surfaces.

1.3.4 The method of FT-IR and preparing QCM analyse

Infrared spectroscopy (IR) shall take place to evaluate the surfaces made by showing different functional groups on the surface specific to the monomers used (HEMA and MAA). Material characterization shall be performed using FT-IR. Initially there will be made a blank background check. Silicon wafers will be used since they are known for their easy character that allows even surfaces and strong binding to form.

Samples of waste water shall be obtained from Kalmar Vatten and sample preparation performed in their laboratory prior to analysis.

System TAMI-FLU Anopore

Membrane REF FILM

REF Nw YES

MIP FILM YES

8 Quartz crystal microbalance studies of oseltamivir-polymer recognition shall be performed using an attana QCM instrument and established protocols. The instrument will be prepared with running buffer, temperature, time and fervency settings and stabilized for one hour before each using setting. Firstly, PBS (Phosphate buffer saline) shall be used at pH 5,1 pH 7,4 and pH 8,5 and five different concentrations of oseltamivir spiked buffer shall be prepared; 10 mg/mL, 5 mg/mL, 2 mg/mL, 1 mg/mL and 0,5 mg/mL for each pH. Afterwards 60 µL of each concentration should be analysed within the QCM. Binding will occur upon the reference chip surfaces inside the QCM. The instrument shall be equilibrated until a stable baseline is observed with a minimum frequency change of 0.5 Hz/min for 5 min at 25 µL/min flowrate.

1.3.5 QCM analyse method

Each time 60 µL of oseltamivir sample will be injected. Before injecting air-bubbles will be removed from the opening of the syringe and a small drop pressed out to get all air from interfering. The syringe will be set up on top of the QCM and injected when curve is stable. The “Load loop” should first be checked as green and right after injection changed over to “Inject sample”-loop. In events each concentration will be noted right away at the notes for the 4th valve at the time of injection. After two minutes of detection the valve should once again be changed back to “Load loop” and the syringe removed and emptied before filled up with a new concentration. All eight samples will be analyzed twice.

1.4 Demarcation Workplan:

General information and supervision

9 Weekly meetings with the supervisory team shall be held to assess progress (Monday afternoons). For questions or assistance of a more practical nature, the supervisory team is available whenever lab work is in progress.

Timeplan

Weeks 1-2*

- project identification and project planning

- familiarization with departmental and lab safety regulations

- introductory seminar on techniques central to project (molecular imprinting and QCM)

Weeks 3-4

- initial reading of prescribed literature concerning:

a) prevalence and risks of Tamiflu/oseltamivir in natural water and waste water systems b) molecular imprinting

c) quartz crystal microbalance technology

- preparation of risk analyses prior to commencement of laboratory work - demonstration of QCM instrumentation

Weeks 5-10

- thin film synthesis

- nano-wire thin film synthesis

- surface characterization (FT-IR and SEM)

Weeks 11-18

- QCM recognition studies (comparison of polymer formats and influence of imprinting effect) - LOD determination, including use of spiked water (from Kalmar Dämme)

- Assessment of samples from Kalmar Vatten (time permitting)

Weeks 19-20

Finalization of written report and oral defense.

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2. Analyse results

2.1 Extraction and H-NMR Analyse

At the second occasion of laboration the 11th _{of mars 2019 a total of 0,675g of oseltamivir were}

processed and purified from nine capsules. After removal of 0,1271 g of water by vacuum-pumping through liquid hydrogen and several extraction steps as described by [17], 0,3734 g of oseltamivir-carboxylase remained.

Figure 2. H-NMR spectrometry diagram describing the pureness of the oseltamivir sample.

2.1.1 FT-IR analyse

11 2.1.2 QCM analyse

Running buffers at pH 5,1 and 7,4 and 8,5 were tried. HEMA EDGMA showed results as supposed but no MAA-chips worked according to plan. The only pH that showed an increase was pH 5,1 in HEMA systems. The oseltamivir solutions at 10 mg/mL and 5 mg/mL did not solubilise in PBS instead only 2 mg/mL down to 0,01 mg/mL where prepared and then used for all further analyzing. MAA FILM did not stabilize in QCM, so no analyzing could even begin within this system.

Each two column in the diagrams stands for a concentration (left to right); 2 mg/mL, 1 mg/mL, 0,5 mg/ml, 0,2 mg/ml, 0,1 mg/ml, 0,05 mg/mL and 0,02 mg/mL.

2.2 QCM Analyse results

Figure 3. Example of QCM-outcome, (HEMA MIP Nw). Two columns per concentration, highest

resonant frequency change at 2 mg/mL and lowest at 0,02 mg/mL. 2.2.1 HEMA QCM analyse results

Each pair of concentrations in the QCM tests got translated into their average frequency change and was translated into a diagram showing frequency and time as a coloured curve. All showing oseltamivir tests except one made with glucose onto a MIP Nw chip.

12 An increase by 1 Hz corresponds to a detection av 0,7 ng contaminant (oseltamivir). A change of 10 Hz up equals 7 ng of oseltamivir.

REF Nw

Figure 4. Frequency change over time, 0-250 sec. Observe that down to 0,02 mg/mL was detected.

These next non coloured diagrams show the same system, but the sensitivity of REF-nanowires. They display only the top frequency for each curve.

Figure 5. Top frequency, around 100 sec for each concentration used in analyse.

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REF Film

Figure 6. Frequency change over time, 0-220 sec. Measures down to 0,05 mg/mL.

Figure 7. Top frequency, around 100 sec for each concentration used in analyse.

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MIP Nw - Glucose

Figure 8. Frequency change over time, 0-150 sec. LOD; 0,02 mg/mL.

Figure 9. Top frequency, >100 sec for each concentration used in analyse except 0,05 mg/mL.

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MIP Nw

Figure 10. Frequency change over time, 0-250 sec. Measurements down to 0,01 mg/mL.

Figure 11. Top frequency, around 100 sec for each concentration used in analyse.

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MIP Film

Figure 12. Frequency change over time, 0-300 sec. Detection down to 0,02 mg/mL.

Figure 13. Top frequency, around 100 sec for each concentration used in analyse.

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Polymer characterization studies

Figure 14. H-NMR-detected functional groups on the different surfaces.

Figure 15. Comparison of detection selectivity between two similar molecules; oseltamivir and

glucose.

Table 3. Highest frequency changes detected for each surface in oseltamivir QCM analyzes.

REF FILM REF Nw MIP FILM MIP Nw

10 <20 30 >80 500 1000 1500 3000 4000 Int ens it y , a. u.

Wavenumber (

)

, cm

Concentration of the analyte (c) , mg/mL Oseltamivir

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3. Discussion

H-NMR spectrometry diagram describing the pureness of the oseltamivir sample permitted further elaborations, see figure 2 in results. Molecularly imprinted polymers with selectivity for oseltamivir as according to the objectives thus was prepared in thin film formats on quartz crystal resonators. Resonators then was used as quartz crystal microbalance recognition elements and used to determine limits of detection considering oseltamivir-spiked water. The reflection absorption infrared spectra (RAIRS) for the MIP and REF films prepared with HEMA and EGDMA monomer in the presence and absence of molecular template, oseltamivir, display similar spectral properties, as they partially should (Fig 14).

In particular bands at 1140 cm-1, 1435 cm-1, 1710 cm-1 and 2927 cm-1 arising from IR active modes, such as n(C-O), d(C-H), n(C=O) and n(C-H), respectively, as expected shows the presence of HEMA and EGDMA moieties in the polymer films.

This data unexpectedly indicate that the presence of oseltamivir template has no apparent influence on the chemical composition of the resultant MIP films.

ship systems did not show any reliable results in QCM however. Probably because MAA-systems were charged negatively and had a repulsive effect on its own nanowires. This could suggestively be solved by using an even lower pH, around 4. In contrast to these inconveniences succeeded diagrams showed the wished out come of a higher frequency peak where an increased concentration pollutant where added into analysis.

19 imprinting since this method shapes a specific molded form for the target molecule to perfectly fit the oseltamivir molecule. Therefor 0,01 mg/mL could not be detected in reference systems. MIPs show a frequency increase more than two times that of references.

None of the systems lowest concentrations (usually 0.02 mg/mL) went over a 2 Hz change in frequency, usually they showed around 1 Hz change. This stresses the fact that the 2 Hz indication at 0,01 mg/mL that was found in MIP Nw is the LOD within the done elaborations. Nonetheless could this be scaled down to 0,1 mg/L with increased loop size (injection at 10x that of 0,01 mg/mL at about 500 µL) and a halved flow rate. It is also all logic that MIP’s will bind the target better due to imprinting and also that the nanowires have the effect of better binding due to a more wide spread surface structure compared to flat films.

In the last diagram it is clearly shown that oseltamivir is easier monitored, or more exactly, has higher affinity by this method then glucose, see figure 14 in results. This has its natural explanation in the fact that the MIP surfaces was imprinted exactly to fit this molecule and not specifically glucose even though it is structured similar to oseltamivir for example.

There was no time permitting further assessment of samples (from Kalmar Vatten) and use of spiked water from Kalmar Dämme do not yet show results other than hundred times too high. The limit that is to be detected is still far away at a real low detection scale that is not possible to detect yet with his method on real samples. An alternative could be nano structuring such as ”polystyrene balls” that possibly could be able to achieve surfaces with even more binding sites needed to detect the LOD searched. This is yet to be examined.

With another nanostructure and polymer types that binds better, have higher affinity and sensitivity at its sensors even lower limits could be monitored further on.

20 not coming down to quantities of µg/L which makes this study relevant in means of comparison between the different water contaminant detecting methods available at this moment.

The timeframe also shows that almost ten years apart they method of HPLC still detects the lowest concentrations of this contaminant.

Nevertheless alternative methods such as for instance nano structuring in form of ”polystyrene balls” as above mentioned are desirable to be examined to find the most affordable way to conquer oseltamivir contaminated water. Also environmental and chemistry students would benefit from taking part of this science and being able to face the challenges together with present scientists within their future professions.

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4. Conclusion

 The presence of oseltamivir template has no apparent influence on the chemical composition of the resultant MIP films.

 Only the HEMA systems stabilized in QCM analyze.

 MAA FILM and Nw did not stabilize in QCM, no analyzing could even begin within that system.

 PBS 5,1 pH was the suitable buffer for a detectible signal in HEMA EDGMA systems.

 Lowest limit of detection (LOD) was at the change of <2 Hz at 0,01 mg/mL and was found in MIP Nw.

 Concentration detection at 0,1 mg/L was able to achieve by increased loop size (injection at 10x that of 0,01 mg/mL at about 500 µL) and a halved flow rate.

 Detections down to µg’s was not possible.

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