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Evaluation of Pseudomonas aeruginosa biofilm formation using piezoelectric tuning fork mass sensors
Sensors and Actuators B 170 (2012) 7–12
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Evaluation of Pseudomonas aeruginosa biofilm formation using piezoelectric tuning fork mass sensors K. Waszczuk a,∗ , G. Gula b , M. Swiatkowski a , J. Olszewski a , W. Herwich a , Z. Drulis-Kawa b , J. Gutowicz b , T. Gotszalk a a b
Faculty of Microsystem Electronics and Photonics, Wroclaw University of Technology, ul. Z. Janiszewskiego 11/17, 50-372 Wroclaw, Poland Institute of Genetics and Microbiology, University of Wroclaw, ul. S. Przybyszewskiego 63/77, 51-148 Wroclaw, Poland
a r t i c l e
i n f o
Article history: Available online 19 November 2010 Keywords: Quartz tuning fork Mass sensor Biofilm Pseudomonas aeruginosa
a b s t r a c t Application of piezoelectric tuning fork mass sensors to bacterial biofilm growth evaluation is presented in this paper. Using low frequency quartz tuning forks as disposable mass sensors, the authors were able to observe various phases of the Pseudomonas aeruginosa biofilm dynamics. A fully electric tuning fork resonance frequency measurement system designed by the authors offers an effective mass measurement resolution of ca. 40 pg. Presented method was sufficient to evaluate the difference between the biofilm formation potential of various P. aeruginosa strains, which is an important factor for the pathogen virulence determination. Results were verified using fluorescent assays and atomic force microscopy visualization. © 2010 Elsevier B.V. All rights reserved.
1. Introduction In many clinical and industrial settings, a biofilm is a cause of hazardous and costly problems. Many of human infections are caused by the biofilm-grown bacteria. It is now well documented that many chronic infections, particularly those involving medical implants and prosthetic devices, involve consortia of bacteria growing as an adherent biofilm enclosed by a polysaccharide matrix [1–4]. Biofilm could be also a source of chemical and toxic metabolite contaminations in water systems and facilitate corrosion of metal structures [5]. In clinical aspect, the most dangerous problems are caused by biofilm-formatting Pseudomonas aeruginosa strains. The P. aeruginosa is a common opportunistic pathogen causing respiratory diseases including cystic fibrosis (CF) and serious nosocomial infections of burn wounds, battlefield injuries, and organ transplant [6]. The monitoring of biofilm development feature is a key step of pathogen virulence determination. Biofilm formation can be detected by several methods including: colorimetric and fluorescent assays (crystal violet staining, resazurin or Syto9 assays), microscopy visualization (scanning electron microscopy (SEM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), atomic force microscopy (AFM)) and fluorescence in situ hybridization [7]. Most of these procedures are time consuming and expensive. The main disadvantage of the mentioned procedures is the lack of precise evaluation of the
∗ Corresponding author. Tel.: +48 71 3203202; fax: +48 71 3283504. E-mail address: [email protected] (K. Waszczuk). 0925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2010.11.019
biofilm progression in a short time. In this work an application of quartz tuning forks in P. aeruginosa biofilm growth monitoring is presented. Crystal oscillators, including quartz tuning forks have been reported to be valuable mass sensors for biochemical and microbiological applications [8–11]. Piezoelectric tuning forks are commonly used for the construction of real time clock (RTC) circuits for battery powered consumer electronic devices. Low resonance frequency resulting in a low power consumption of oscillators based on tuning forks allowed these crystal resonators to dominate the market of portable devices containing an RTC such as wrist watches, portable music players, etc. They are also present in RTC circuits of personal computers main boards. These BT cut quartz resonators show a superior resonant frequency stability in the room temperature range to standard AT cut quartz crystal microbalances (QCM) and can be easily integrated into the measurement system without designing a complicated holder. High quality factor, in the range of 105 for a tuning fork oscillating in air, results in a high resolution of the resonant frequency measurement. Due to a very low price, mass produced tuning forks can be used as disposable mass sensors, which is a very important feature for biochemical and microbiological applications. 2. Materials and methods 2.1. Piezoelectric tuning fork as a biofilm mass sensor In contrary to standard quartz crystal microbalances, which operate as thickness-shear resonators, the effective mass of molecules adhered to the quartz tuning fork surface is not equal
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K. Waszczuk et al. / Sensors and Actuators B 170 (2012) 7–12
to their real mass. Nevertheless, the real mass of bacterial biofilm grown on the tuning fork can be estimated on the basis of the tuning fork resonant frequency shift. However, it has to be assumed that the concentration of cells in the biofilm matrix is constant over the sensor surface. This is justified as the adhesion of bacterial cells to the tuning fork surface is a random process. It is also assumed that due to very high value of the tuning fork spring constant (ca. 30,000 N/m), the surface stress influence on the resonance frequency can be ignored. The effective mass of a molecule adhered to the tuning fork surface can be calculated using the formula: mmolecule eff = mmolecule (lx /l)3 [12,13], where l is the tuning fork beam length and lx is the distance between the adhered molecule and the beam clamping area. Considering an infinitely small part of the biofilm matrix, placed on the infinitely short part of the sensor beam, Eq. (1) can be formed. mbiofilm
eff
3
lx l mbiofilm l l
=
⇔
dmbiofilm
eff
dl
=
3
lx 1 · mbiofilm l l
(1) mbiofilm is the overall actual mass of the bacterial biofilm covering the sensor and mbiofilm eff is its effective mass. Through the integration of the above function the relation between the effective and the real mass of the biofilm can be obtained, see Eq. (2).
mbiofilm
eff
l
=
mbiofilm
l 3
l
l
0
l
=
mbiofilm
0
l4
x
dlx
3
· lx dlx =
Fig. 1. (a) Commercial quartz tuning forks and (b) Au ball bound to the tuning fork surface at the beam end.
mbiofilm l4
·
1 4 1 l = mbiofilm 4 4
(2) 2.2. Tuning fork resonant frequency measurement
Sensitivity of the tuning fork mass sensor, defined as the resonator effective mass change divided by the mechanical resonant frequency shift caused by the tuning fork loading can be calculated on the basis of the tuning fork geometry and physical properties of quartz [11], as in Eq. (3). Knowing the relation between the overall effective and the overall actual mass of the biofilm covering the sensors beams, the authors calculated the sensitivity of a tuning fork used as biofilm mass sensor – Eq. (3). meff f
≈ ⇔
Et 3 w 8l3 ˘ 2 f03 mbiofilm f
≈ 40 ng/Hz,
mbiofilm
≈ 160 ng/Hz
f
eff
=
1 mbiofilm · 4 f (3)
l, w, t are, respectively the length, width and thickness of the tuning fork, f0 is the tuning fork resonance frequency and E is the Young modulus of quartz. Calculated value of meff /f has been verified using the Cleveland method [14]. Tuning fork was loaded using 200 m Au balls at the end of its beam (Fig. 1b), then the resonant frequency shift was measured. The sensitivity of 44 ng/Hz obtained experimentally showed a 11% variation versus the meff /f value calculated analytically on the basis of tuning fork dimensions measured using an optical microscope. Such sensitivity, together with the achieved 10 mHz resolution of resonant frequency measurement, results in a 400 pg effective mass measurement resolution for 3.7 mm long standard quartz tuning forks used for experiments. In more demanding applications effective mass measurement resolution of 40 pg can be obtained using 1.8 mm long commercial quartz tuning forks (Fig. 1a). Calculated mass of the biofilm grown on the sensor was normalized versus the sensor surface (Fig. 6). This gives a much better insight into the bacteria cells concentration and can be easily compared to results obtained using other mass sensitive resonant devices such as standard QCMs, membranes, cantilevers, etc.
Various methods of tuning fork resonant frequency measurement have been reported: self-excited generator circuit [9,11], systems with mechanical actuation [11,15], systems based on tuning fork admittance measurement [16]. Self-excited circuits react to the resonance frequency shift with a very low time constant, however, no information about other oscillation parameters is obtained. Systems using a mechanical excitation are additionally capable of measuring oscillations amplitude and a quality factor of a piezoelectric oscillator. Systems based on admittance modulus measurement have all advantages of systems with mechanical excitation but they are fully electric, thus not requiring additional mechanical actuator. Tuning fork admittance versus frequency characteristics show a very good correlation with an amplitude of mechanical oscillations measured as a function of actuation signal frequency (Fig. 2). Multi-channel tuning fork admittance modulus versus frequency measurement system has been designed for the purpose of presented experiments (Fig. 3). 1 mHz stability of actuation signal, which is crucial for the tuning fork resonant frequency measurement resolution is guaranteed with a dedicated 8-channel generator, using a direct digital signal synthesis (DDS), clocked from an oven controlled 20 MHz crystal oscillator (OCXO). Multi-channel transimpedance amplifier has been integrated in the hermetic measurement chamber, which can contain up to 8 sensors. Voltage signal proportional to the tuning fork current is demodulated using a dedicated, digitally controlled 8-channel RMS-DC converter and then recorded using a National Instruments 6251 16 bit data acquisition card. 2.3. Bacterial strains The P. aeruginosa ATCC 27853 and P. aeruginosa PAO1 (ATCC 15692) strains were used as a model of a biofilm-grown bacteria. As a negative control, a non biofilm-forming bacteria Escherichia coli
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9
Fig. 2. Tuning fork admittance modulus and the displacement of the end of a tuning fork beam versus frequency of a 60 mV actuation signal.
ATCC 25922 strain was applied. All strains were purchased from the American Type Culture Collection (ATCC). Bacteria were stored at −70 ◦ C in Trypticase Soy Broth (Becton Dickinson and Company, Cockeysville, MD, USA) supplemented with 20% glycerol. For the experiments strains were refreshed on Muller Hinton Agar (MHA) (Becton Dickinson and Company, Cockeysville, MD, USA) for 18 h. 2.4. Bacterial biofilm formation on tuning forks For the experiments, refreshed bacterial strains were diluted in a physiological saline (PS) to an optical density equal to the McFarland No. 0.5. Then the culture was suspended in 2 ml of Muller Hinton Broth (MHB) (Becton Dickinson and Company, Cockeysville, MD, USA) in 24 wells titrate plate to obtain final density of 5 × 105 cells per ml (cfu/ml) in each well. Sterilized tuning forks soldered in dedicated holders (Fig. 4b) were suspended in wells containing the bacterial culture. The plates were then incubated for 24 h at 37 ◦ C. After every hour of the experiment 6 tuning forks were taken out and rinsed by sterile water for 20 min. Rinsed resonators were fixed in absolute alcohol for 30 min and dried for 10 min in a laminar air flow chamber. Each tuning fork was then placed in the hermetic measurement chamber (Fig. 4a). The encased measurement chamber was taken out of the laminar air flow chamber and connected to the measurement system. Resonant frequency shift of each tuning fork was measured to estimate a mass of the bacterial biofilm adhered to the sensors surface. All tests were repeated at least 3 times per strain. Simultaneously, bacterial growth in titrate plate
Fig. 4. (a) Hermetic measurement chamber with an integrated 8-channel transimpedance preamplifier and (b) quartz tuning fork soldered in a dedicated holder.
well was determined by serial dilution and bacterial colony count on MHA plates. 2.5. Biofilm formation and quantification by crystal violet (CV) assay The dynamics of adhesion and biofilm formation was also measured by standard biomass assays (based on the quantification of matrix and both living and dead cells) using crystal violet (CV) [17]. For the experiments, refreshed bacterial strains were diluted in a physiological saline to an optical density equal to the McFarland No. 0.5. Then the culture was suspended in MHB and 200 l of suspension were added into a round-bottomed polystyrene 96well microtiter plate to obtain final density of 5 × 105 cfu/ml in each well. The plates were then incubated for 24 h at 37 ◦ C. After every 2 h the supernatant containing planktonic (free-floating) non-adhered cells was removed from each well and plates were rinsed using 200 l of physiological saline and 125 l of 0.01% CV solution was added to each well. After 20 min the excess CV was removed by washing the plates twice using 200 l of physiological saline. Finally, bound CV was released by adding 200 l of 96% ethanol (Sigma–Aldrich Chemie GmbH, Steinheim, Germany) and 100 l of suspension were transferred to new plate. The absorbance was measured at 595 nm using a multilabel microtiter plate reader (UVM 340, AsysHitech). Positive control consisted of 100 l of 0.01% CV solution. Negative controls consisted of 96% ethanol. All assays were repeated at 16 times per strain. 2.6. AFM visualization The presence of bacterial cells adhered onto the surface of quartz tuning forks was monitored by a microscopy visualization using an atomic force microscope. Sensors were rinsed in distilled water to remove the non-adherent forms of bacteria, then prepared by fixing in an absolute alcohol for 30 min and then examined using a Nanoman VS microscope with NanoScope V controller (Veeco Instruments, Santa Barbara, CA, USA). 3. Results and discussion
Fig. 3. Block diagram of the multi channel system for tuning fork admittance modulus measurement.
The biofilm development proceeds in three major steps: (i) attachment of the planktonic cell forms to the surface; (ii) matura-
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Fig. 7. P. aeruginosa ATCC 27853, P. aeruginosa PAO1 and E. coli ATCC 25922 culture growth curve. Fig. 5. The dynamics of bacteria cell adhesion and biofilm formation measured by standard biomass assays using crystal violet (CV).
tion of a biofilm structure, (iii) partial degradation of biofilm, release and dispersal of sessile cells [18]. In presented experiments the dynamics of adhesion and biofilm formation was measured independently using standard colorimetric biomass assay (Fig. 5) and using piezoelectric tuning forks as biofilm mass sensors (Fig. 6). The quantification of a biofilm matrix and both living and dead bacterial cells (Fig. 5) showed that E. coli ATCC 25922 strain as a non biofilm forming bacteria exhibited 2–3% of CV adsorption. The curve of P. aeruginosa biomass development presented characteristic biofilm formation features: (i) attachment of the planktonic cell (1–6 h of incubation); (ii) biofilm maturation (6–12 h of incubation), (iii) dispersion (after 12 h of incubation). The peak of dye adsorption was noticed between 10 h and 14 h of incubation and reached the values of 36% and 60% for P. aeruginosa ATCC 27853 and P. aeruginosa PAO1, respectively. After 24 h of the experiment the biomass amount of P. aeruginosa biofilm dropped down to about one third of the highest level. The measurement of biofilm development using tuning forks exhibited similar results (Fig. 6). No changes of sensors resonant frequency in case of the E. coli ATCC 25922 culture was noted. The same biofilm formation steps in the same time interval as in CV assay could be seen for P. aeruginosa strains. The P. aeruginosa PAO1 strain produced biofilm structure more intensively in comparison to P. aeruginosa ATCC 27853 reaching 70 Hz and 30 Hz reduction
Fig. 6. The dynamics of bacteria cell adhesion and biofilm formation measured using piezoelectric tuning fork mass sensors.
of resonant frequency, respectively. The maximum calculated surface mass density reached a value of 80 pg/102 m2 in case of the P. aeruginosa PAO1 and 30 pg/102 m2 for P. aeruginosa ATCC 27853. For both applied methods the phase of P. aeruginosa cell attachment to the surface and the phase of the biofilm formation were noted in equal time intervals. The obtained results were controlled by standard growth curve determination presented in Fig. 7. All three bacterial culture multiplied exhibiting classical growth curve characteristics. Before the third hour of the incubation adaptation of the culture to the fresh medium could be seen. Between 2 h and 7 h the exponential phase took place, where the culture doubled until the nutrients were present in the medium (the number of cells grew in time exponentially). At the end of the exponential phase of the culture growth maturation of P. aeruginosa biofilm occurred (Figs. 5 and 6). In stationary phase of the growth curve (after 7 h of incubation), as essential nutrients were depleted or toxic products increased, the number of new cells was almost equal to the number of dying cells. The rate of culture growth slowed down (Fig. 7), but the biofilm mass still rose up until the 12th hour of the experiment (Figs. 5 and 6). The P. aeruginosa PAO1 produced the biofilm intensively and after the 12th hour of incubation a sudden decrease of biomass and number of cells was noticed in the culture (the death phase). In case of P. aeruginosa ATCC 27853 strain the degradation of biofilm was correlated with further growth of the culture which could mean that acquired nutrients were obtained from the matrix disintegration. Colonies of biofilm formatting bacteria were placed randomly on the tuning fork surface and visible as stains of various size in the optical microscope image. AFM microscopy visualization was used to confirm the presence of bacterial cells adhered to the sensors surface (Fig. 8a and b). The structures visible in AFM images had the width of around 0.6 m and the length of around 1.6 m which are typical values for P. aeruginosa cells. During the biofilm maturation phase (9th hour of the experiment) the height of bacterial structure was in the range of 25–50 nm, which shows the presence of large amount of polysaccharide encasing P. aeruginosa cells. After the biofilm dispersion phase (24th hour of the experiment) the measured height of P. aeruginosa structure equalled their width (Fig. 8b), which means that the polysaccharide matrix was no more present in the observed bacterial colonies. This shows that the rapid mass reduction during the biofilm dispersion phase is not only due to the increase of planktonic cells of P. aeruginosa at the expense of the number of bacteria in the biofilm matrix but is also an effect of the reduction of polysaccharide matrix in the nutrients deprived environment. The presented mass measurement system is a precise and efficient tool for monitoring of bacterial biofilm growth. It could be
K. Waszczuk et al. / Sensors and Actuators B 170 (2012) 7–12
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Fig. 8. Topography of P. aeruginosa ATCC 27853 colonies grown on a tuning fork surface measured using an AFM after the 9th hour of the experiment (a) and the 24th hour of the experiment (b).
applied to fast evaluation of antimicrobials activity in elimination and prevention of biofilm formation. The use of piezoelectric tuning forks with a functionalised surface could be also helpful for fast evaluation of the suitability of chemical compounds for fabrication of anti-biofilm and anti-adhesive layers. 4. Conclusions Commercial piezoelectric tuning forks were applied as disposable biofilm mass sensors. Presented technique is cost effective and requires only a relatively simple sensor preparation. A mass surface density of the biofilm was estimated during adhesion, maturation and dispersion phases of P. aeruginosa ATCC 27853 and P. aeruginosa PAO1 biofilm formation. The biofilm formation potential of these two bacterial strains was compared. The P. aeruginosa PAO1 and the P. aeruginosa ATCC 27853 biofilm achieved a maximum mass surface density of 80 pg/102 m2 and 30 pg/102 m2 , respectively. AFM measurements confirmed the presence of biofilm on sensors surface and were useful to observe the state of polysaccharide matrix in the biofilm. Results showed good correlation with the information obtained using standard colorimetric biomass assay and confirmed the utility of this technique for the pathogen virulence determination. Acknowledgements This work was financed by the project: Detectors and sensors for measuring factors hazardous to environment – modeling and monitoring of threats, POIG.01.03.01-02-002/08-00. We would like to acknowledge Piotr Paletko and Krzysztof Nieradka for performing AFM scans of the biofilm matrix, Michal Gorowij for the help in preparation of tuning forks for the experiments, Rafal Niedziela for designing the measurement chamber,
Joanna Majewska for performing the biofilm colorimetric biomass assays and Grzegorz Wielgoszewski for proof reading the article.
References [1] J.W. Costerton, R.T. Irwin, K.J. Cheng, The bacterial glycocalyx in nature and disease, Annual Reviews of Microbiology 35 (1981) 299–324. [2] A.G. Gristina, C.D. Hobgood, L.X. Webb, Q.N. Myrvik, Adhesive colonization of biomaterials and antibiotic resistance, Biomaterials 8 (1987) 423–426. [3] J.C. Nickel, J. Heaton, A. Morales, J.W. Costerton, Bacterial biofilm in persistent penile prosthesis-associated infection, Journal of Urology 135 (1986) 586–588. [4] J.H. Tenney, M.R. Moody, K.A. Newman, S.C. Schimpff, J.C. Wade, J.W. Costerton, Adherent microorganisms on lumenal surfaces of long-term intravenous catheters. Importance of Staphylococcus epidermidis in patients with cancer, Archives of Internal Medicine 146 (1986) 1949–1954. [5] N.J. Dowling, M.W. Mittelman, J.C. Danko, Microbially Influenced Corrosion and Biodeterioration, The University of Tennessee Press, 1990. [6] T.B. May, D. Shinabarger, R. Maharaj, J. Kato, L. Chu, J.D. DeVault, S. Roychoud´ A. Berry, A.K. Rothmel, T.K. Misraand, A.M. Chakrabarty, hury, N.A. Zielinski, Alginate synthesis by Pseudomonas aeruginosa: a key pathogenic factor in chronic pulmonary infections of cystic fibrosis patients, Clinical Microbiology Reviews 4 (1991) 191–206. [7] C. Hanning, M. Follo, E. Hellwig, A. Al-Ahmad, Visualization of adherent microorganisms using different techniques, Journal of Medical Microbiology 59 (2010) 1–7. [8] M.Z. Atashbar, B. Bejcek, A. Vijh, S. Singamaneni, QCM biosensor with ultra thin polymer film, Sensors and Actuators B: Chemical 107 (2005) 945–951. [9] X. Su, C. Dai, J. Zhang, S.J. O’Shea, Quartz tuning fork biosensor, Biosensors and Bioelectronics 17 (2002) 111–117. [10] V. Reipa, J. Almeida, K.D. Cole, Long-term monitoring of biofilm growth and disinfection using a quartz crystal microbalance and reflectance measurements, Journal of Microbiological Methods 66 (2006) 449–459. [11] J. Zhang, S. O’Shea, Tuning forks as micromechanical mass sensitive sensors for bio- or liquid detection, Sensors and Actuators: B 94 (2003) 65–72. [12] R. Porksch, Nondestructive Added Mass Spring Calibration with the MFP-3D, Ph.D., Asylum Reasearch, Santa Barbara (2003). [13] S. Dohn, R. Sandberg, W. Svendsen, A. Boisen, Enhanced functionality of cantilever based mass sensors using higher modes, Applied Physics Letters 86 (2005) 233501. [14] J.P. Cleveland, S. Manne, D. Bocek, P.K. Hansma, A non-destructive method for determining the spring constant of cantilevers for scanning force microscopy, Review of Scientific Instruments 64 (1993) 403.
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[15] W.H.J. Rensen, N.F. van Hulst, S.B. Kammer, Imaging soft samples in liquid with tuning fork based shear force microscopy, Applied Physics Letters 77 (2000) 1557–1559. [16] P. Westerhoff, N. Tao, N. Guzman, K.C. Kruger, F. Tsow, E. Forzani, Microfabricated Tuning Fork Based Sensor for Disinfection By-products in Drinking Water, Proof of Concept, Arizona State University, 2008. [17] G.D. Christensen, W.A. Simpson, J.J. Younger, L.M. Baddour, F.F. Barrett, D.M. Melton, E.H. Beachey, Adherence of coagulase negative staphylococci to plastic tissue culture plates: a quantitative model for the adherence of staphylococci to medical devices, Journal of Clinical Microbiology 22 (1985) 996–1006. [18] H.C. Flemming, J. Wingender, The biofilm matrix, Nature Reviews Microbiology 9 (2010) 623–633.
Biographies Karol Waszczuk was born in 1984 in Wodzislaw Slaski, Poland. He graduated from the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology in 2007, receiving the MSc degree in electronics and telecommunications. Since then, he has been working at Research and Development Department for EBS Inkjet Systems Poland. He is pursuing his PhD degree at Wroclaw University of Technology, working on biochemical and microbiological applications of resonant mass sensors. Grzegorz Gula was born in 1984 in Sroda Slaska, Poland. He graduated from the Faculty of Biology Science at University of Wroclaw in 2008, receiving the MSc degree in biology with a specialization in microbiology. Currently he is pursuing his PhD degree at the Department of Physicochemistry of Microorganisms at University of Wroclaw working on application of new methods of observation of bacterial biofilm formation. Michal Swiatkowski was born in 1983 in Wrocław, Poland. He graduated from the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology in 2007, receiving the MSc degree in electronics and telecommunications. Currently, he is a PhD student at the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology, working on applications of quartz crystal resonators in biosensing. Jarosław Olszewski was born in 1985 in Wrocław, Poland. He graduated from the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology in 2009, receiving the MSc degree in electronics and telecommunications.
For year and half he worked at The Division of Micro- and Nanostructures Metrology at the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology, providing software and hardware support in field of biochemical sensing. Currently he is working at Nokia Siemens Networks, improving his software development skills in embedded systems. Wiktor Herwich was born in 1984 in Walcz, Poland. He graduated from the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology in 2008. His research interests involve piezoelectric rezonators and their applications as mass sensors. Zuzanna Drulis-Kawa was born in Wroclaw, Poland. She received the MSc and PhD degrees of biology from University of Wroclaw, Department of Nature Science in 1994 and 2000, respectively. She received first and second clinical microbiology degrees at Medical University of Wroclaw in 1997 and 2000, respectively. She has been honored with bioMerieux-PTM research award (2009) and the prize of Prof. K. Bassalik Contest (2006) for her scientific work. She is the head of the Department of Pathogen’s Biology and Immunology in Institute of Genetics and Microbiology at University of Wroclaw, Poland. She has authored over 60 scientific publications. Jan Gutowicz was born in Walbrzych, Poland. In 1971 he received his MSc degree in chemistry from the Faculty of Mathematics, Physics and Chemistry of University of Wroclaw. In 1997 he received the PhD degree in biophysics from the Wroclaw University of Medicine. He has been honored with award of Polish Academy of Sciences for his scientific research, the prize of Polish Ministry of Science and Higher Education for the academic handbook and, many Rector prizes of University of Wroclaw for his scientific and educational records. He is the Head of the Department of Physico-Chemistry of Microorganisms at the Institute of Genetics and Microbiology of University of Wroclaw. He has authored or co-authored over 80 scientific publications. Teodor Pawel Gotszalk was born in Wroclaw, Poland. He received the MSc degrees from the Faculties of Electronics and of Electrical Engineering of Wroclaw University of Technology in 1989 and 1991, respectively. In 1996, he received the PhD degree from the Institute of Electronic Technology of the Wroclaw University of Technology. He has been honored with Siemens research award (2000) and the prize of Polish Science Foundation FNP (1997) for his scientific work. He is the head of the Division of Micro- and Nanostructures Metrology at the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology. He has authored over 100 scientific publications.
Contents lists available at ScienceDirect
Sensors and Actuators B: Chemical journal homepage: www.elsevier.com/locate/snb
Evaluation of Pseudomonas aeruginosa biofilm formation using piezoelectric tuning fork mass sensors K. Waszczuk a,∗ , G. Gula b , M. Swiatkowski a , J. Olszewski a , W. Herwich a , Z. Drulis-Kawa b , J. Gutowicz b , T. Gotszalk a a b
Faculty of Microsystem Electronics and Photonics, Wroclaw University of Technology, ul. Z. Janiszewskiego 11/17, 50-372 Wroclaw, Poland Institute of Genetics and Microbiology, University of Wroclaw, ul. S. Przybyszewskiego 63/77, 51-148 Wroclaw, Poland
a r t i c l e
i n f o
Article history: Available online 19 November 2010 Keywords: Quartz tuning fork Mass sensor Biofilm Pseudomonas aeruginosa
a b s t r a c t Application of piezoelectric tuning fork mass sensors to bacterial biofilm growth evaluation is presented in this paper. Using low frequency quartz tuning forks as disposable mass sensors, the authors were able to observe various phases of the Pseudomonas aeruginosa biofilm dynamics. A fully electric tuning fork resonance frequency measurement system designed by the authors offers an effective mass measurement resolution of ca. 40 pg. Presented method was sufficient to evaluate the difference between the biofilm formation potential of various P. aeruginosa strains, which is an important factor for the pathogen virulence determination. Results were verified using fluorescent assays and atomic force microscopy visualization. © 2010 Elsevier B.V. All rights reserved.
1. Introduction In many clinical and industrial settings, a biofilm is a cause of hazardous and costly problems. Many of human infections are caused by the biofilm-grown bacteria. It is now well documented that many chronic infections, particularly those involving medical implants and prosthetic devices, involve consortia of bacteria growing as an adherent biofilm enclosed by a polysaccharide matrix [1–4]. Biofilm could be also a source of chemical and toxic metabolite contaminations in water systems and facilitate corrosion of metal structures [5]. In clinical aspect, the most dangerous problems are caused by biofilm-formatting Pseudomonas aeruginosa strains. The P. aeruginosa is a common opportunistic pathogen causing respiratory diseases including cystic fibrosis (CF) and serious nosocomial infections of burn wounds, battlefield injuries, and organ transplant [6]. The monitoring of biofilm development feature is a key step of pathogen virulence determination. Biofilm formation can be detected by several methods including: colorimetric and fluorescent assays (crystal violet staining, resazurin or Syto9 assays), microscopy visualization (scanning electron microscopy (SEM), transmission electron microscopy (TEM), confocal laser scanning microscopy (CLSM), atomic force microscopy (AFM)) and fluorescence in situ hybridization [7]. Most of these procedures are time consuming and expensive. The main disadvantage of the mentioned procedures is the lack of precise evaluation of the
∗ Corresponding author. Tel.: +48 71 3203202; fax: +48 71 3283504. E-mail address: [email protected] (K. Waszczuk). 0925-4005/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.snb.2010.11.019
biofilm progression in a short time. In this work an application of quartz tuning forks in P. aeruginosa biofilm growth monitoring is presented. Crystal oscillators, including quartz tuning forks have been reported to be valuable mass sensors for biochemical and microbiological applications [8–11]. Piezoelectric tuning forks are commonly used for the construction of real time clock (RTC) circuits for battery powered consumer electronic devices. Low resonance frequency resulting in a low power consumption of oscillators based on tuning forks allowed these crystal resonators to dominate the market of portable devices containing an RTC such as wrist watches, portable music players, etc. They are also present in RTC circuits of personal computers main boards. These BT cut quartz resonators show a superior resonant frequency stability in the room temperature range to standard AT cut quartz crystal microbalances (QCM) and can be easily integrated into the measurement system without designing a complicated holder. High quality factor, in the range of 105 for a tuning fork oscillating in air, results in a high resolution of the resonant frequency measurement. Due to a very low price, mass produced tuning forks can be used as disposable mass sensors, which is a very important feature for biochemical and microbiological applications. 2. Materials and methods 2.1. Piezoelectric tuning fork as a biofilm mass sensor In contrary to standard quartz crystal microbalances, which operate as thickness-shear resonators, the effective mass of molecules adhered to the quartz tuning fork surface is not equal
8
K. Waszczuk et al. / Sensors and Actuators B 170 (2012) 7–12
to their real mass. Nevertheless, the real mass of bacterial biofilm grown on the tuning fork can be estimated on the basis of the tuning fork resonant frequency shift. However, it has to be assumed that the concentration of cells in the biofilm matrix is constant over the sensor surface. This is justified as the adhesion of bacterial cells to the tuning fork surface is a random process. It is also assumed that due to very high value of the tuning fork spring constant (ca. 30,000 N/m), the surface stress influence on the resonance frequency can be ignored. The effective mass of a molecule adhered to the tuning fork surface can be calculated using the formula: mmolecule eff = mmolecule (lx /l)3 [12,13], where l is the tuning fork beam length and lx is the distance between the adhered molecule and the beam clamping area. Considering an infinitely small part of the biofilm matrix, placed on the infinitely short part of the sensor beam, Eq. (1) can be formed. mbiofilm
eff
3
lx l mbiofilm l l
=
⇔
dmbiofilm
eff
dl
=
3
lx 1 · mbiofilm l l
(1) mbiofilm is the overall actual mass of the bacterial biofilm covering the sensor and mbiofilm eff is its effective mass. Through the integration of the above function the relation between the effective and the real mass of the biofilm can be obtained, see Eq. (2).
mbiofilm
eff
l
=
mbiofilm
l 3
l
l
0
l
=
mbiofilm
0
l4
x
dlx
3
· lx dlx =
Fig. 1. (a) Commercial quartz tuning forks and (b) Au ball bound to the tuning fork surface at the beam end.
mbiofilm l4
·
1 4 1 l = mbiofilm 4 4
(2) 2.2. Tuning fork resonant frequency measurement
Sensitivity of the tuning fork mass sensor, defined as the resonator effective mass change divided by the mechanical resonant frequency shift caused by the tuning fork loading can be calculated on the basis of the tuning fork geometry and physical properties of quartz [11], as in Eq. (3). Knowing the relation between the overall effective and the overall actual mass of the biofilm covering the sensors beams, the authors calculated the sensitivity of a tuning fork used as biofilm mass sensor – Eq. (3). meff f
≈ ⇔
Et 3 w 8l3 ˘ 2 f03 mbiofilm f
≈ 40 ng/Hz,
mbiofilm
≈ 160 ng/Hz
f
eff
=
1 mbiofilm · 4 f (3)
l, w, t are, respectively the length, width and thickness of the tuning fork, f0 is the tuning fork resonance frequency and E is the Young modulus of quartz. Calculated value of meff /f has been verified using the Cleveland method [14]. Tuning fork was loaded using 200 m Au balls at the end of its beam (Fig. 1b), then the resonant frequency shift was measured. The sensitivity of 44 ng/Hz obtained experimentally showed a 11% variation versus the meff /f value calculated analytically on the basis of tuning fork dimensions measured using an optical microscope. Such sensitivity, together with the achieved 10 mHz resolution of resonant frequency measurement, results in a 400 pg effective mass measurement resolution for 3.7 mm long standard quartz tuning forks used for experiments. In more demanding applications effective mass measurement resolution of 40 pg can be obtained using 1.8 mm long commercial quartz tuning forks (Fig. 1a). Calculated mass of the biofilm grown on the sensor was normalized versus the sensor surface (Fig. 6). This gives a much better insight into the bacteria cells concentration and can be easily compared to results obtained using other mass sensitive resonant devices such as standard QCMs, membranes, cantilevers, etc.
Various methods of tuning fork resonant frequency measurement have been reported: self-excited generator circuit [9,11], systems with mechanical actuation [11,15], systems based on tuning fork admittance measurement [16]. Self-excited circuits react to the resonance frequency shift with a very low time constant, however, no information about other oscillation parameters is obtained. Systems using a mechanical excitation are additionally capable of measuring oscillations amplitude and a quality factor of a piezoelectric oscillator. Systems based on admittance modulus measurement have all advantages of systems with mechanical excitation but they are fully electric, thus not requiring additional mechanical actuator. Tuning fork admittance versus frequency characteristics show a very good correlation with an amplitude of mechanical oscillations measured as a function of actuation signal frequency (Fig. 2). Multi-channel tuning fork admittance modulus versus frequency measurement system has been designed for the purpose of presented experiments (Fig. 3). 1 mHz stability of actuation signal, which is crucial for the tuning fork resonant frequency measurement resolution is guaranteed with a dedicated 8-channel generator, using a direct digital signal synthesis (DDS), clocked from an oven controlled 20 MHz crystal oscillator (OCXO). Multi-channel transimpedance amplifier has been integrated in the hermetic measurement chamber, which can contain up to 8 sensors. Voltage signal proportional to the tuning fork current is demodulated using a dedicated, digitally controlled 8-channel RMS-DC converter and then recorded using a National Instruments 6251 16 bit data acquisition card. 2.3. Bacterial strains The P. aeruginosa ATCC 27853 and P. aeruginosa PAO1 (ATCC 15692) strains were used as a model of a biofilm-grown bacteria. As a negative control, a non biofilm-forming bacteria Escherichia coli
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Fig. 2. Tuning fork admittance modulus and the displacement of the end of a tuning fork beam versus frequency of a 60 mV actuation signal.
ATCC 25922 strain was applied. All strains were purchased from the American Type Culture Collection (ATCC). Bacteria were stored at −70 ◦ C in Trypticase Soy Broth (Becton Dickinson and Company, Cockeysville, MD, USA) supplemented with 20% glycerol. For the experiments strains were refreshed on Muller Hinton Agar (MHA) (Becton Dickinson and Company, Cockeysville, MD, USA) for 18 h. 2.4. Bacterial biofilm formation on tuning forks For the experiments, refreshed bacterial strains were diluted in a physiological saline (PS) to an optical density equal to the McFarland No. 0.5. Then the culture was suspended in 2 ml of Muller Hinton Broth (MHB) (Becton Dickinson and Company, Cockeysville, MD, USA) in 24 wells titrate plate to obtain final density of 5 × 105 cells per ml (cfu/ml) in each well. Sterilized tuning forks soldered in dedicated holders (Fig. 4b) were suspended in wells containing the bacterial culture. The plates were then incubated for 24 h at 37 ◦ C. After every hour of the experiment 6 tuning forks were taken out and rinsed by sterile water for 20 min. Rinsed resonators were fixed in absolute alcohol for 30 min and dried for 10 min in a laminar air flow chamber. Each tuning fork was then placed in the hermetic measurement chamber (Fig. 4a). The encased measurement chamber was taken out of the laminar air flow chamber and connected to the measurement system. Resonant frequency shift of each tuning fork was measured to estimate a mass of the bacterial biofilm adhered to the sensors surface. All tests were repeated at least 3 times per strain. Simultaneously, bacterial growth in titrate plate
Fig. 4. (a) Hermetic measurement chamber with an integrated 8-channel transimpedance preamplifier and (b) quartz tuning fork soldered in a dedicated holder.
well was determined by serial dilution and bacterial colony count on MHA plates. 2.5. Biofilm formation and quantification by crystal violet (CV) assay The dynamics of adhesion and biofilm formation was also measured by standard biomass assays (based on the quantification of matrix and both living and dead cells) using crystal violet (CV) [17]. For the experiments, refreshed bacterial strains were diluted in a physiological saline to an optical density equal to the McFarland No. 0.5. Then the culture was suspended in MHB and 200 l of suspension were added into a round-bottomed polystyrene 96well microtiter plate to obtain final density of 5 × 105 cfu/ml in each well. The plates were then incubated for 24 h at 37 ◦ C. After every 2 h the supernatant containing planktonic (free-floating) non-adhered cells was removed from each well and plates were rinsed using 200 l of physiological saline and 125 l of 0.01% CV solution was added to each well. After 20 min the excess CV was removed by washing the plates twice using 200 l of physiological saline. Finally, bound CV was released by adding 200 l of 96% ethanol (Sigma–Aldrich Chemie GmbH, Steinheim, Germany) and 100 l of suspension were transferred to new plate. The absorbance was measured at 595 nm using a multilabel microtiter plate reader (UVM 340, AsysHitech). Positive control consisted of 100 l of 0.01% CV solution. Negative controls consisted of 96% ethanol. All assays were repeated at 16 times per strain. 2.6. AFM visualization The presence of bacterial cells adhered onto the surface of quartz tuning forks was monitored by a microscopy visualization using an atomic force microscope. Sensors were rinsed in distilled water to remove the non-adherent forms of bacteria, then prepared by fixing in an absolute alcohol for 30 min and then examined using a Nanoman VS microscope with NanoScope V controller (Veeco Instruments, Santa Barbara, CA, USA). 3. Results and discussion
Fig. 3. Block diagram of the multi channel system for tuning fork admittance modulus measurement.
The biofilm development proceeds in three major steps: (i) attachment of the planktonic cell forms to the surface; (ii) matura-
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Fig. 7. P. aeruginosa ATCC 27853, P. aeruginosa PAO1 and E. coli ATCC 25922 culture growth curve. Fig. 5. The dynamics of bacteria cell adhesion and biofilm formation measured by standard biomass assays using crystal violet (CV).
tion of a biofilm structure, (iii) partial degradation of biofilm, release and dispersal of sessile cells [18]. In presented experiments the dynamics of adhesion and biofilm formation was measured independently using standard colorimetric biomass assay (Fig. 5) and using piezoelectric tuning forks as biofilm mass sensors (Fig. 6). The quantification of a biofilm matrix and both living and dead bacterial cells (Fig. 5) showed that E. coli ATCC 25922 strain as a non biofilm forming bacteria exhibited 2–3% of CV adsorption. The curve of P. aeruginosa biomass development presented characteristic biofilm formation features: (i) attachment of the planktonic cell (1–6 h of incubation); (ii) biofilm maturation (6–12 h of incubation), (iii) dispersion (after 12 h of incubation). The peak of dye adsorption was noticed between 10 h and 14 h of incubation and reached the values of 36% and 60% for P. aeruginosa ATCC 27853 and P. aeruginosa PAO1, respectively. After 24 h of the experiment the biomass amount of P. aeruginosa biofilm dropped down to about one third of the highest level. The measurement of biofilm development using tuning forks exhibited similar results (Fig. 6). No changes of sensors resonant frequency in case of the E. coli ATCC 25922 culture was noted. The same biofilm formation steps in the same time interval as in CV assay could be seen for P. aeruginosa strains. The P. aeruginosa PAO1 strain produced biofilm structure more intensively in comparison to P. aeruginosa ATCC 27853 reaching 70 Hz and 30 Hz reduction
Fig. 6. The dynamics of bacteria cell adhesion and biofilm formation measured using piezoelectric tuning fork mass sensors.
of resonant frequency, respectively. The maximum calculated surface mass density reached a value of 80 pg/102 m2 in case of the P. aeruginosa PAO1 and 30 pg/102 m2 for P. aeruginosa ATCC 27853. For both applied methods the phase of P. aeruginosa cell attachment to the surface and the phase of the biofilm formation were noted in equal time intervals. The obtained results were controlled by standard growth curve determination presented in Fig. 7. All three bacterial culture multiplied exhibiting classical growth curve characteristics. Before the third hour of the incubation adaptation of the culture to the fresh medium could be seen. Between 2 h and 7 h the exponential phase took place, where the culture doubled until the nutrients were present in the medium (the number of cells grew in time exponentially). At the end of the exponential phase of the culture growth maturation of P. aeruginosa biofilm occurred (Figs. 5 and 6). In stationary phase of the growth curve (after 7 h of incubation), as essential nutrients were depleted or toxic products increased, the number of new cells was almost equal to the number of dying cells. The rate of culture growth slowed down (Fig. 7), but the biofilm mass still rose up until the 12th hour of the experiment (Figs. 5 and 6). The P. aeruginosa PAO1 produced the biofilm intensively and after the 12th hour of incubation a sudden decrease of biomass and number of cells was noticed in the culture (the death phase). In case of P. aeruginosa ATCC 27853 strain the degradation of biofilm was correlated with further growth of the culture which could mean that acquired nutrients were obtained from the matrix disintegration. Colonies of biofilm formatting bacteria were placed randomly on the tuning fork surface and visible as stains of various size in the optical microscope image. AFM microscopy visualization was used to confirm the presence of bacterial cells adhered to the sensors surface (Fig. 8a and b). The structures visible in AFM images had the width of around 0.6 m and the length of around 1.6 m which are typical values for P. aeruginosa cells. During the biofilm maturation phase (9th hour of the experiment) the height of bacterial structure was in the range of 25–50 nm, which shows the presence of large amount of polysaccharide encasing P. aeruginosa cells. After the biofilm dispersion phase (24th hour of the experiment) the measured height of P. aeruginosa structure equalled their width (Fig. 8b), which means that the polysaccharide matrix was no more present in the observed bacterial colonies. This shows that the rapid mass reduction during the biofilm dispersion phase is not only due to the increase of planktonic cells of P. aeruginosa at the expense of the number of bacteria in the biofilm matrix but is also an effect of the reduction of polysaccharide matrix in the nutrients deprived environment. The presented mass measurement system is a precise and efficient tool for monitoring of bacterial biofilm growth. It could be
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Fig. 8. Topography of P. aeruginosa ATCC 27853 colonies grown on a tuning fork surface measured using an AFM after the 9th hour of the experiment (a) and the 24th hour of the experiment (b).
applied to fast evaluation of antimicrobials activity in elimination and prevention of biofilm formation. The use of piezoelectric tuning forks with a functionalised surface could be also helpful for fast evaluation of the suitability of chemical compounds for fabrication of anti-biofilm and anti-adhesive layers. 4. Conclusions Commercial piezoelectric tuning forks were applied as disposable biofilm mass sensors. Presented technique is cost effective and requires only a relatively simple sensor preparation. A mass surface density of the biofilm was estimated during adhesion, maturation and dispersion phases of P. aeruginosa ATCC 27853 and P. aeruginosa PAO1 biofilm formation. The biofilm formation potential of these two bacterial strains was compared. The P. aeruginosa PAO1 and the P. aeruginosa ATCC 27853 biofilm achieved a maximum mass surface density of 80 pg/102 m2 and 30 pg/102 m2 , respectively. AFM measurements confirmed the presence of biofilm on sensors surface and were useful to observe the state of polysaccharide matrix in the biofilm. Results showed good correlation with the information obtained using standard colorimetric biomass assay and confirmed the utility of this technique for the pathogen virulence determination. Acknowledgements This work was financed by the project: Detectors and sensors for measuring factors hazardous to environment – modeling and monitoring of threats, POIG.01.03.01-02-002/08-00. We would like to acknowledge Piotr Paletko and Krzysztof Nieradka for performing AFM scans of the biofilm matrix, Michal Gorowij for the help in preparation of tuning forks for the experiments, Rafal Niedziela for designing the measurement chamber,
Joanna Majewska for performing the biofilm colorimetric biomass assays and Grzegorz Wielgoszewski for proof reading the article.
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Biographies Karol Waszczuk was born in 1984 in Wodzislaw Slaski, Poland. He graduated from the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology in 2007, receiving the MSc degree in electronics and telecommunications. Since then, he has been working at Research and Development Department for EBS Inkjet Systems Poland. He is pursuing his PhD degree at Wroclaw University of Technology, working on biochemical and microbiological applications of resonant mass sensors. Grzegorz Gula was born in 1984 in Sroda Slaska, Poland. He graduated from the Faculty of Biology Science at University of Wroclaw in 2008, receiving the MSc degree in biology with a specialization in microbiology. Currently he is pursuing his PhD degree at the Department of Physicochemistry of Microorganisms at University of Wroclaw working on application of new methods of observation of bacterial biofilm formation. Michal Swiatkowski was born in 1983 in Wrocław, Poland. He graduated from the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology in 2007, receiving the MSc degree in electronics and telecommunications. Currently, he is a PhD student at the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology, working on applications of quartz crystal resonators in biosensing. Jarosław Olszewski was born in 1985 in Wrocław, Poland. He graduated from the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology in 2009, receiving the MSc degree in electronics and telecommunications.
For year and half he worked at The Division of Micro- and Nanostructures Metrology at the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology, providing software and hardware support in field of biochemical sensing. Currently he is working at Nokia Siemens Networks, improving his software development skills in embedded systems. Wiktor Herwich was born in 1984 in Walcz, Poland. He graduated from the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology in 2008. His research interests involve piezoelectric rezonators and their applications as mass sensors. Zuzanna Drulis-Kawa was born in Wroclaw, Poland. She received the MSc and PhD degrees of biology from University of Wroclaw, Department of Nature Science in 1994 and 2000, respectively. She received first and second clinical microbiology degrees at Medical University of Wroclaw in 1997 and 2000, respectively. She has been honored with bioMerieux-PTM research award (2009) and the prize of Prof. K. Bassalik Contest (2006) for her scientific work. She is the head of the Department of Pathogen’s Biology and Immunology in Institute of Genetics and Microbiology at University of Wroclaw, Poland. She has authored over 60 scientific publications. Jan Gutowicz was born in Walbrzych, Poland. In 1971 he received his MSc degree in chemistry from the Faculty of Mathematics, Physics and Chemistry of University of Wroclaw. In 1997 he received the PhD degree in biophysics from the Wroclaw University of Medicine. He has been honored with award of Polish Academy of Sciences for his scientific research, the prize of Polish Ministry of Science and Higher Education for the academic handbook and, many Rector prizes of University of Wroclaw for his scientific and educational records. He is the Head of the Department of Physico-Chemistry of Microorganisms at the Institute of Genetics and Microbiology of University of Wroclaw. He has authored or co-authored over 80 scientific publications. Teodor Pawel Gotszalk was born in Wroclaw, Poland. He received the MSc degrees from the Faculties of Electronics and of Electrical Engineering of Wroclaw University of Technology in 1989 and 1991, respectively. In 1996, he received the PhD degree from the Institute of Electronic Technology of the Wroclaw University of Technology. He has been honored with Siemens research award (2000) and the prize of Polish Science Foundation FNP (1997) for his scientific work. He is the head of the Division of Micro- and Nanostructures Metrology at the Faculty of Microsystems Electronics and Photonics of Wroclaw University of Technology. He has authored over 100 scientific publications.