Elsevier

Thin Solid Films

Volume 517, Issue 15, 1 June 2009, Pages 4299-4302
Thin Solid Films

Comparative study on Pulsed Laser Deposition and Matrix Assisted Pulsed Laser Evaporation of urease thin films

https://doi.org/10.1016/j.tsf.2008.11.141Get rights and content

Abstract

Urease thin films were produced by Matrix Assisted Pulsed Laser Evaporation (MAPLE) and Pulsed Laser Deposition from two types of targets: frozen water solutions of urease with different concentrations (1–10% m/v) and pure urease pellets. The fluence of the ablating KrF excimer laser was varied between 300 and 2200 mJ/cm2. Fourier transform infrared spectra of the deposited films showed no difference as compared to the original urease. Morphologic studies proved that the films consist of a smooth “base” layer with embedded micrometer-sized droplets. Absorption-coefficient measurements contradicted the traditional “absorptive matrix” model for MAPLE deposition. The laser energy was absorbed by urease clusters leading to a local heating-up and evaporation of the frozen matrix from the uppermost layer accompanied by the release of dissolved urease molecules. Significant enzymatic activity of urease was preserved only during matrix assisted transfer.

Introduction

Biosensing devices are of increasing importance in research, biology, medical science, environmental protection and even food industry. Production of biosensors often requires immobilization of different active biomaterials in form of thin films on the appropriate transducer devices. Pulsed Laser Deposition (PLD) found to be effective in preparation of inorganic thin layers was proven to be hardly applicable for deposition of complex organic materials, especially the sensitive biomaterials, where – if applicable – low laser fluences, near to ablation threshold have to be applied resulting in very low deposition rates [1], [2], [3], [4]. To avoid the photonic and thermal damages of these materials during the thin film deposition the Matrix Assisted Pulsed Laser Evaporation (MAPLE) technique was developed, where the target material is embedded in an absorptive volatile matrix. MAPLE has been used for deposition of organic and biomaterial thin layers such as carbohydrates, biopolymers, proteins, etc. [5], [6], [7], [8], [9], [10].

Urease is an enzyme frequent in biological systems where it catalyses the hydrolysis of urea. It is related to protein intake, nitrogen metabolism and excretion as follows:(NH2)2CO+3H2OUREASEHCO3+2NH4++OH

The development of a sensor of urea based on this catalytic reaction is of considerable interest in clinical chemistry, since the nitrogen concentration of the human serum in the form of urea is a measure of the kidney function. Urea analysis also finds application in agro-food chemistry and environmental monitoring. Different types of urease-based biosensors were developed based on the ion production of this catalytic reaction using ion-sensitive field effect transistors [11], [12], ion selective [13], [14] or pH sensitive electrodes [15]. György et al. studied the enzymatic activity of urease thin films deposited by MAPLE [16].

In this work we present our comparative study on the deposition of urease thin films by pulsed laser based methods (MAPLE and PLD) by ablating with KrF excimer laser beam two types of targets: frozen water solutions of urease with different concentrations and pure urease pellets.

Section snippets

Experimental details

Urease enzyme (from jack beans, ≥ 5 U/mg, Merck) solved in ultra pure water in different concentrations (1%, 2%, 3.5%, 5% and 10% m/v, respectively) and frozen at − 30 °C was used as target material for MAPLE deposition. The maximum applied concentration was 10% because in case of higher concentrations the urease was not completely dissolved. For comparison of the two deposition methods thin films were deposited also by conventional PLD method using pellets pressed from urease powder at 440 MPa

Fourier-transformed infrared spectroscopy

As a first step the dependence of the IR spectra on the laser fluence was studied at 10% target concentration in 300–2200 mJ/cm2 fluence range. At lower fluences practically no deposition was observed and the ice slowly sublimed from the target leaving behind the urease in a sponge-like structure. As Fig. 1. shows in the studied range no significant changes of the spectra as compared to the reference could be observed, with except of the Amide I band around 1640 cm 1 that showed a significant

Discussions

The results obtained by PLD can be explained by photochemical and thermal effect of the laser irradiation. Although, beside the nanometer sized particulates micrometer sized grains of materials (containing target material protected from direct light absorption) are also transferred to the substrate, these are probably affected by the strong thermal effect of the laser irradiation during PLD (the denaturation temperature of the urease enzyme is around 90 °C). Generally, the effectiveness of the

Summary

Thin films were deposited with conventional PLD and MAPLE deposition from urease using as targets pressed pellets and frozen different concentration solutions, respectively. Significant enzymatic activity of the deposited material was only observed when applying the MAPLE method. Absorption coefficient measurements indicated that when using water solvent the idea of absorbing matrix does not stand, i.e. the relatively high absorptivity of matrix is not a general requirement of a successful

Acknowledgements

The authors acknowledge the support of a Hungarian–Romanian Governmental Bilateral Agreement “Biosensors design using laser radiation” 2006–2007, Hungarian Scientific Research Foundation OTKA (K 67818) National Office for Research and Technology-Hungary NKTH (OMFB 00345/2006), Romanian Ministry for Education and Research (CEEX 150/2006).

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