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Tannerella forsythia is among the most potent triggers of periodontal diseases, and approaches to understand underlying mechanisms are currently intensively pursued. A similar to 22-nm-thick, 2D crystalline surface (S-) layer that completely covers Tannerella forsythia cells is crucially involved in the bacterium-host cross-talk. The S-layer is composed of two intercalating glycoproteins (TfsA-GP, TfsB-GP) that are aligned into a periodic lattice. To characterize this unique S-layer structure at the nanometer scale directly on intact T. forsythia cells, three complementary methods, i.e., small-angle X-ray scattering (SAXS), atomic force microscopy (AFM), and single-molecular force spectroscopy (SMFS), were applied. SAXS served as a difference method using signals from wild-type and S-layer-deficient cells for data evaluation, revealing two possible models for the assembly of the glycoproteins. Direct high-resolution imaging of the outer surface of T. forsythia wild-type cells by AFM revealed a p4 structure with a lattice constant of similar to 9.0 nm. In contrast, on mutant cells, no periodic lattice could be visualized. Additionally, SMFS was used to probe specific interaction forces between an anti-TfsA antibody coupled to the AFM tip and the S-layer as present on T. forsythia wild-type and mutant cells, displaying TfsA-GP alone. Unbinding forces between the antibody and wild-type cells were greater than with mutant cells. This indicated that the TfsA-GP is not so strongly attached to the mutant cell surface when the co-assembling TfsB-GP is missing. Altogether, the data gained from SAXS, AFM, and SMFS confirm the current model of the S-layer architecture with two intercalating S-layer glycoproteins and TfsA-GP being mainly outwardly oriented. Copyright (c) 2013 John Wiley & Sons, Ltd.

This paper describes the usage of a portable Surface Plasmon Resonance system for label free detection of DNA from Mycobacterium tuberculosis complex. A single-strand oligodeoxynucleotide, carrying a thiol end group (and also a spacer arm), which is complementary of the target characteristic sequence of Mycobacterium tuberculosis complex was used as the "probe" and immobilized onto the gold coated surfaces of the Surface Plasmon Resonance slides as Nano-overlayers by using a direct immobilization method based on the self-assembled over-layer formation. A modulator-spacer molecule 6-mercapto-1-hexanol was also co-immobilized in order to optimize the surface density of the probe-oligodeoxynucleotides and to orient them for better interaction. It is demonstrated that the reported Surface Plasmon Resonance biosensor platform can be used successfully to detect target molecules both synthetic and Polymerase Chain Reaction products very effectively at nM levels. The sensor platforms can also be regenerated with 2.5 mM HCl and reused effectively. It has been shown that developed biosensor has high specificity, sensitivity, sensor reproducibility and long shelf life for both small synthetic oligodeoxynucleotides and Polymerase Chain Reaction amplified real samples.

ÖZET Sunulan çalışmanın amacı piezoelektrik kristalli mikrobalans sistemine dayanan bir nükleik asit sensörü geliştirmektir. 10 MHz salınım frekanslı kuvartz kristaller kullanılmıştır. Kristal yüzeyleri önce bir plazma polimerizasyon (`elektriksel boşalım`) sisteminde etilen diamin plazmasıyla etkileştirilmiştir. Etkileştirme süresi 1-5 dak aralığında değiştirilmiş, boşalım gücü ve monomer akış hızı ise sırasıyle 10 watt ve 25-30 ml/dak'da sabit tutulmuştur. Kristal yüzey modifikasyonundan sonra oluşan kristalin salınım frekansındaki kaymalar bir mikrobalans sistemiyle ölçülmüştür. En uygun plazma etkileştirme süresi 2.5 dak olarak bulunmuştur. Plazma polimerizasyon basamağından sonra ölçülen ortalama frekans kayması 143±24 Hz'dir. İkinci basamakta, plazma polimerizasyonuyla kristal yüzeylerde oluşturulan primer amin grupları, kristaller glutaraldehit (GA) ile etkileştirilerek aldehit gruplarına dönüştürülmüştür. GA konsantrasyonu ve inkübasyon süresi değiştirilirken, ortam sıcaklığı 4°C'de sabit tutulmuştur. Burada optimum GA konsantrasyonu ve inkübasyon süresi sırasıyla %2.5 ve 2 saat olarak bulunmuştur. Bu basamaktan sonra ölçülen ortalama frekans kayması 88±13 Hz'dir. Çift sarmal oligonükleotid ile plazma polimerizasyonuyla modifîye edilen kristaller farklı pH ve iyonik güçte çözeltilerde bulunan çift sarmal oligonükleotid ile etkileştirilmiştir. Oligonükleotid adsorpsiyonu pH:10.6 ve iyonik güç 0.1 iken en düşük bulunmuştur. GA-modifiye krsital yüzeylere oligonükleotid immobilizasyonu farklı koşullarda çalışılmıştır. Birinci grupta, oligonükleotid konsantrasyonu değiştirilirken (0.5-4.0 ug/ml), immobilizasyon süresi sabit 3 saat olarak sabit tutulmuştur. En yüksek immobilizasyon 1.0 fig/ml konsantrasyonunda bulunmuştur. İkinci gruptaki deneylerde oligonükleotid konsantrasyonu 1.0 o.g/ml olarak sabit tutulmuş, immobilizasyon 1-22 saat aralığında değiştirilmiştir. 3 saatlik süreden sonra immobilizayon miktarında önemli bir değişiklik olmadığı gözlenmiştir. İmmobilizasyon sonrası ölçülen ortalama frekans kayması 126±12 Hz'dir. Son adımda yüzeye kovalent olarak bağlı olmayan oligonükleotid sarmalı yüzeyden dehibridize edilmiştir (uzaklaştırılmıştır). Kovalent bağlı oligonükleotid tek sarmalınıVI taşıyan nükleik asit sensörü hibridizasyon deneylerinde kullanılmıştır. Burada komplementer sarmalın konsantrasyonu (0.00 l-l ng/ml) ve etkileştirme süresi (1-25 dak) değiştirilmiştir. Komplementer sarmalın ortamdaki konsantrasyonunun artışı ile ölçülen frekans kaymasının arttığı gözlenmiştir. Buna karşın 5 dak lık süreden daha uzun sürelerde hibridizasyonda önemli değişiklik olmadığı bulunmuştur. Burada ölçülen ortalama frekans kayması 43.5±9 Hz'dir. Taramalı tünelleme mikroskobu ile yapılan görüntülemelerde her adımdan sonra yüzey değişikleri izlenmiştir. Anahtar Kelimeler: Nükleik asit sensörleri; piezoelektrik kuvars kristalleri; plazma polimerizasyonu; glutraldehit etkileştirilmesi; oligonükleotid immobilizasyonu; hibridizasyon/ dehibridizasy on vn ABSTRACT The aim of this study is to develop a nucleic asit sensor based on piezoelectric crystal microbalance system. Quartz crystals having 10 MHz resonans frequency have been used as the sensing element. Crystal surfaces were first treated with ethylene diamine (EDA) plasma in a plasma polymerization (`glow-discharge`) system. The treatment time was changed between 1-5 min., but the discharge power and the monomer flow rate were kept constant, 15 watt and 25-30 ml/min.c, respectively. A microbalance system was used to measure the shifts in the resonance frequency of the crystals after the surface treatment. The optimal treatment time was 2.5 min. The average frequency shift after the plazma polymerization step was 143±24 Hz. In the second step, the primary amine groups on the crystal surfaces obtained after plazma polymerization were converted to aldehyde groups by treating the surfaces with glutaraldehyde (GA). The GA concentration and the incubation time were changed while the treatment temperature was constant at 4°C. The optimal GA concentration and incubation time were 2.5% and 2 h, respectively. The average frequency shift after this treatment was 88±13 Hz. The plazma-modified crystals were treated with aqueous solutions (at different pH and ionic strenght) of the double strand oligonucleotide. The non-specific oligonucleotide adsorption was found minimum at pH: 1 0.6 and an ionic strength of 0. 1. Oligonucleotide immobilization onto the GA-modified crystal surafaces was investigated at different conditions. In the first group, only the oligonucleotid concentration was changed (0.5-4.0 ug/ml), in which the immobilization time was 3 h. The maximum immobilization was achieved at the concentration of 1.0 ug/ml. In the second group, the oligonucleotid concentration was 1.0 ug/ml, while the immobilization time was changed in the range of 1-22 h. No significant increase in the immobilization was observed after 3 h. The average frequency shift after immobilization was 126±12 Hz. At the last step, one of the strands (not covalently bound to the surface) was dehybridized (removed). The nucleic acid sensor carrying the covalently bound single strand was then used in the hybridization experiments. The concentration of the complementary strand and the incubation time was changed in these tests. The frequencyvııı shifts were increased when the concentration of the complementary strand was increased. There was no significant changed in the hybridization after 5 min incubation time. The average frequency shift measured was 43.5±9 Hz. The images obtained with the scanning tunneling micrscope revealed the changes on the surfaces after each treatment. Keywords: Nucleic acid sensors; piezoelectric quartz crystals; plazma polymerization, glutaraldehyde treatment; Oligonucleotide immobilization; Hybridization/ dehybridization 72