Postgraduate symposium 2011 Biosensors and chelator nanoparticles for groundwater radionuclide detection

1. Postgraduate symposium 2011Biosensors and chelator nanoparticles for groundwater radionuclide detection David J.R. Conroy Supervisor: Prof Paul Millner Co-supervisor: Dr Doug Stewart DIAMOND University Research Consortium, funded by the EPSRC IMSB FACULTY OF BIOLOGICAL SCIENCES Millner labs

2. Introduction to Biosensors

3. Overview

4. Detection and analysis method for molecular analytes

5. Background to the PhD and application of biosensors

6. Background to nuclear legacy waste and contamination problems Automated remote monitoring of the long-term performance of civil engineering structures using robust, durable sensors is now routine. Groundwater monitoring still relies heavily on labour intensive sampling and laboratory testing to directly determine contaminant concentrations. The high cost of monitoring acts against adopting otherwise very cost-effective but long-term passive remediation options, such as bio-stimulation. Additionally slow data turn-round time for traditional sampling and analysis makes process control during remediation very difficult.

7. Proposed system

8. Analytes of Interest

9. Surface layer protein JG-A12 for binding UO22+ • Proteins as metal ion receptor components • Bacterial and Archaeouter membrane coating, purpose unknown other than cell structure support, low molecular weight sieving and protection from cytolysis. • B.sphaericus JG-A12 evolved in radioactive waste piles to survive and bind uranium (working with Pollmann labs, Dresden). • Protein identified purified and grown for use as a potential biosensor recognition element. (Figure from Pollmann 2005)

10. Biosensor assembly (II): Short molecular linkers • BIOCONJUGATION ON GOLD • Direct bioconjugation removes the large capacitance base from the mSAM with a porous membrane structure. Lateral shifting and aggregation is removed creating a lower interfering resistance component signal. • Sulfo-SMCC binds to amine groups of a self assembled 4-ATP monolayer; it’s maleimide groups bond to free sulfhydryl cysteine groups on the SLP. • Currently this system gives a much more sensitive and reproducible response to analyte binding. Linker length approx 1.5nm, analyte binding disrupts electrical double layer, capacitance based signal. Schematic of protein binding to molecular tethers on a gold surface.

11. Interrogating biosensor and analyte binding in real time:Electrochemical Impedance Spectroscopy Impedance spectroscopy is essentially the observed capacitance and resistance phase change observed on an applied potential with frequency. • Frequency response analysis of bioconjugated electrodes to uranyl ion binding shows sensitivity at lower frequencies (imaginary impedance response with frequency of s-layer conjugated electrode in increasing uranyl nitrate concentrations • 10 kHz, • 1 kHz, • 100 kHz, • 10 Hz, • 1 Hz •0.1 Hz). 0 V applied vsAgAgCl, 10mM PBS supporting electrolyte, 51 points calculated 0.01V rms. Low frequency sensitivity of biosensor interface. Low frequency corresponds to mass transport and analyte delivery. High frequency interactions show reaction kinetics.

12. Interrogating sensors using Electrochemistry • Electrochemistry is the study of chemical reactions under imposed electrical fields. Faradaic electrochemistry traditionally uses electron transfer and redox couples while the non-faradaic uses capacitance based systems. • We use a combination of the two. • Electrochemical cell contains 3 electrodes: • A current or potential is applied between the working and counter and measured againstthereference electrode. • Various materials trialled as the working electrode. Figure showing typical 3 electrode cell set up with bubbler, stirrer and faraday cage. Working: Gold, platinum, carbon, mercury Reference: Ag/AgCl Counter: Platinum

13. Uranyl response Greater specificity to uranyl compounds independent of compound. Binds linear +6 oxidation state UO22+. pH cycling between 3 and 10 shows –Z” decrease and recovery suggesting desorption – reversible binding. Deaeration with argon prior and during trials minimises oxygen and co2 interference. Lines show response from different sensors of different production and protein batches over several months. Real time capacitance response to analytes. Sensors were exposed to increasing concentrations of analyte and stirred continuously for 15 mins before a 30 min equilibration period EIS scans were performed in 10 mM PBS at 0V vs Ag /AgCl at a perturbation of 10 mV. (A) – Response of biosensor to different uranyl compound response (nUranyl nitrate on 6 hr old electrode, luranyl nitrate response from a 7 d old electrode, undepleted uranyl nitrate response ‚uranyl acetate response). The data shows no differentiation between uranyl compounds as all are able to bind with the UO22+ in the +6 oxidation state.

14. Interfering analyte ions Cobalt, caesium, cadmium and nickel repeats show some non specific binding but average a 10 to 20% lower than uranyl. Response of biosensor to a range of interfering divalent cations. (l average uranium response for comparison, n nickel nitrate, caesium sulphate, ‚ cadmium nitrate, ¿cobalt chloride.) calcium and magnesium have little response as protein in already saturated with these ions.

15. X-ray photoelectron spectroscopy confirmed protein binding site blocking and sequential layer deposition of biosensor construction • Individual element analysis. Significant carbon ratio increase in the modified S-layer biosensors supporting successful blocking of carboxylates and phosphates. (A) (B) (C) (A) SEM image of organic SLP biosensor layer bound to a gold working electrode. Dense protein layer covalently bound with boundaries between protein domains. (B) XPS analysis of surface composition of top 5nm of bound protein layer before (— ) and after (----) chemical modification of phosphate and carboxy groups. The data show a 30.2% carbon C 1s to gold 4f peak ratio increase confirming successful modification of analyte binding sites. (C) XPS layer by layer biosensor deposition showing increased carbon to gold ratio.

16. Optimisation and prototype development: Commercial and bespoke flow cell. Commercial ceramic based substrates with carbon or gold inks. Single or dual working electrode setup to a circular single channel methacrylate flow cell hooked up to syringe or peristaltic driving force.

17. Chemosensors using crown ethers and cavitands Detection via frequency dissipation as a function of mass

18. Supra molecular structures and Chemosensors for Sr-90 and Cs-137

19. Aqueous analyte Ionic liquid layer loaded with DC18C6 host Electrode Chemosensor interrogation How to determine analyte binding with chemosensors? Impedance of a host covered layer, binding blocks central cavity creating a significant change in current / capacitance etc. Oxidation or reduction (ASV) of bound host analyte at an electrode – diffusional based systems eg solvent extraction / immiscible interfaces (right figure). Often crown ethers are tethered to a chromophore, which is quenched upon binding – an expensive one shot optical based system. Mass based detection of analyte binding to host via frequency dissipation as a function of mass.

20. First successful crown ether host synthesis confirmed by NMR and mass spectroscopy NMR analyses  field spin of active nuclei in a magnetic field to determine new bond generation [M+H]+ ofAza-18 crown 6 Successful product expected at 679.54 g /mol Cholesterol aza-18C6 Triethylamine Cholesterol chloroformate Aza 18C6 DMF contamination 74 Triethylamine 102 NMR: Chloroform-d, 99.8% (Isotopic), contains 0.03% v/v TMS, 300MHz tube Deuterium oxide 99.9 atom % D, contains 0.05% 3-(trimethylsilyl)propionic-2,2,3,3-d4 acid, sodium salt, 300Mhz tube . Mass spec: Chloroform dispersion from solvent recrystalisation. (

21. Second host – a hydrophobic tail tethered crown ether First synthesised a control molecule to compare to proposed caesium binding host. Similar resorcin[4]arene that lacks the specificity for caesium for use in parallel control experiments. 2,c-8,c-14,c-20-tetraundecyl-4,6,10,12,16,18,22,24-octahydroxyresorc[4]arene A 4 aromatic ring with 8 oxygen groups on the upper rim and 4 C11H23 hydrocarbon tails on the lower rim.

22. Quartz crystal micro balance bindingresor[4]calixarene into mSAM MHDA • Successful host molecule insertion. • Repeatable strontium binding and removal by use of metal chelator. • Not concentration specific enough. • Mass decrease rather than increase. (I) dH2O; (ii) Ethanol; (iii)10/mg/ml resorcalixarene in acetone; (iv) ethanol; (v) dH2O; (vi) 1mM Strontium nitrate; (vii) dH2O; (viii) 1mM EDTA; (ix) dH2O; (x) 1mM Strontium nitrate (xi) dH2O; (xii) EDTA.

23. Functionalised metal affinity surfaces and nanoparticles Silica nanoparticles functionalised with metal chelators

24. High density metal chelators Due to a lack of other binding proteins a combination of traditional scintilation counting on high affinity surfaces is being used. Nitrilioacetic acid (NTA) interacts with a metal ion through 4 bonds. Sulfonates interact with metals via 3 oxygen and a sulphur atom. ETDA – a pentadentate molecule, will coordinate 5 bonds with the metal ligand.

25. Glass substrates Glass substrates left to right: 190 gsm cloth , glass tissue, 290 gsm cloth glass fibre

26. Secondary ion mass spec on sulphonate surface (5% carboxysilane on aluminium 3% dH2O) • Blank aluminium – Positive scans showed Al+ (27), possible Na+ (39) salt contamination. • Negative scans O- (16),OH- (17) ,AlO2- (59) • (B) Sulfonate tethered layer. • Positive scans showed Al (27) Si (26) C5H3+ (69). • Negative scans showed C2H-, CH3O- organic fragments. • Lower aluminium peaks in (B) showing organic layer. (A) (B)

27. EDTA surface (1 step deposition on to a pre hydroxl coated surface) Microscope image of aluminium surface imaged, (B) Triplicate FT-IR transmission mode scans of three different areas per sample. Sample prepared as 5% EDTA-silane reacted in 2% dH2O ethanol as a 6h surface condensation reaction to AlOH surface layer.

28. Sulfonate deposition (2 step) • Microscope image of aluminium surface imaged, (B) Triplicate FT-IR transmission mode scans of three different areas per sample. Sample prepared as 5% carboxysilane reacted in 2% dH2O ethanol 6h, EDACed to sulfonate. • Taurine (sulfonate containing molecule) peptide bonded to a carboxylate coated substrate.

29. %T 4000.0 3800 3600 3400 3200 3000 2800 2600 2400 2200 2000 1900 1800 1700 1600 1500 1400 1300 1200 1100 1000 900 800 750.0 cm-1 Scaled comparison of sulfonate surfaces on aluminium . Peaks scaled to an equal aluminium ratio showing the higher transmittance of the wsc/sulfo NHS linkage showing a higher density of bound sulfonates. 6h 5% carboxy silane 3% dH2O in ethanol sulfo NHS+ EDACed to taurine in MES 2h reaction (red), NTA(green). Optimised sulfonate linkage Water soluble carbodiimide and zero length cross linker creates higher density deposition

30. Functionalised metal affinity nanoparticles Gold, silver and silica nanoparticles functionalised with metal chelators

31. Functionalised metallic nanoparticles (gold and silver) Colloidal suspensions can be created by steric, polymeric or charge based particle stabilisation. 10mM chloroauric acid only compared to 5 increasing stabiliser concentrations in duplicate (left to right: No stabiliser, 10uM, 1mM, 10mM, 0.1M, 0.5M) Oleylamine (1-Amino-9-octadecene) Long surfactant like alkene molecule used as an acid and reducing agent on HAuCl3

32. Surface Silication theory Bioconjugatable silane compounds typically consist of a central silicon atom bonded to an alkyl linker arm (CH2)n linked to the functional or reactive group. The most common coupling groups being methoxy (alkyl H-C chain linked to an O); alkoxyls, or ethyl ether, the ethoxyl silanes. These groups are hydrolysable and allow direct attachment to hydroxyl coated surfaces.

33. Silica nano, micro and macro particles Functionality wanted reacted with base molecule with growth in three dimensions 1.TEOS tetraethyl orthosilicate 2. TMOS Tetramethylorthosilicate

34. Silica nano, micro and macro particles 1.TEOS tetraethyl orthosilicate 2. TMOS TetramethylOrthosilicate

35. Solvent experiments – altering nucleation rates Reactions based on 5% silane in organic solvent + 3% H2O. Theory based on altering hydrophobicity and polarity of environment (silanes initially insoluble in water until they hydrolyse), altering apolarity effects phase exchange and possibility the size of nanoparticles. Solvents used: methanol, ethanol, DMS, DMSO, acetone, water

36. Time and heat based reaction • Up to 50oC bimodal particles generated. • Beyond 60oC generates slight surface layer coating. • Above 80oC silanes come out of liquid phase and generate bulk polymer coatings. • 8h reaction hydrolysis optimal Figure - Room temperature based reaction 5% TEMOS 3% dH2O in ethanol pH 5 with carboxysilane.

37. Conclusions and ongoing work Prototype uranyl biosensor shown with a degree of specificity and sensitivity a head of any other available system. Now being transferred to portable usable device with flow cell design. Lifetime trials. Moving onto more complex analytes and synthetic ground water. Alternative hosts. The biosensor impedance technologies described has potential for bespoke systems using specific receptors. Working on B53 and B57 strains. Scintillation based methods. High surface area silicated sorbents and nanoparticles with traditional chelating groups with specificity delivered from decay signature. Loading functionalised nanoparticles on support matrices.

38. Thanks to: • Supervisors • Prof Paul Millner and Dr Doug Stewart • Millner labs: • Dr Alex Vakourov, Dr Tim Gibson, • Rebecca Caygill, Muhammad AshrafShahidan, David Conroy, Jim Tiernan, Natalie Hirst. • Pollmannlabs for S-layer JG-A12 protein supply. • Publications: • Conroy, D.J.; Millner, P.A.; Stewart, D.I.; Pollmann, K. Biosensing for the Environment and Defence: Aqueous Uranyl Detection Using Bacterial Surface Layer Proteins. Sensors2010, 10, 4739-4755.

39. Molarity to ppm or ppb