Investigation of Polar and Nonpolar Cyclotides Separation from Violet Extract Through Microfluidic Chip

Cyclotides (CTs) as a cyclic peptide obtained from different groups of plants have been very attractable field of research for scientists because of their specific properties like their natural function as host defense agents. CTs are bioactive peptides from plants that characterized by their head-to-tail cyclic backbone and knotted arrangement of their three conserved disulfide bonds. Their natural function is thought to be as host defense agents and a single plant can express dozens to hundreds of CTs. CTs stand out as a family of antimicrobial peptides (AMPs) because of their exceptional stability, structural plasticity, unique biochemical target, and Gram-negative selective antimicrobial action. These features together with recent advancements in the methods of production of CTs make them an intriguing prospect from a drug development perspective. To accomplish this aim, as part of a separation, detection and research of anti-cancer properties CTs study, we investigate the separation of cyclotides in violets into polar and non-polar groups by microfluidic chips

Investigation of the effect of channel structure and flow rate on on-chip bacterial lysis

Successful lysis of cells/microorganisms is a key step in the sample preparation in fields like molecular biology, bioengineering, and biomedical engineering. This study therefore aims to investigate the lysis of bacteria on-chip and its dependence on both microfluidic channel structure and flow rate. Effects of temperature on lysis on-chip were also investigated. To perform these investigations, three different microfluidic chips were designed and produced (straight, zigzag and circular configurations), while the length of the channels were kept constant. As an exemplary case, Mycobacterium smegmatis was chosen to represent the acid-fast bacteria. Bacterial suspensions of 1.5 McFarland were injected into the chips at various flow rates (0.6-8 ll/min) either at room temperature or 500 C. In order to understand the on-chip lysis performance fully, off-chip experiments were carried out at durations which are equal to those bacteria spent in the channel from inlet to the outlet at different flow rates. We also performed COMSOL multiphysics program simulations to evaluate further the effect of the applied parameters. As a result, we found that the structure and the flow rate do not affect lysis over all in all investigated channel types, however on-chip experiments at room temperature produced more effective lysis compared to the on-chip and the off-chip samples performed at higher temperatures. Interestingly on-chip experiments at higher tempratures do not result in effective lysis

Label-free molecular detection of antibiotic susceptibility for Mycobacterium smegmatis using a low cost electrode format

Today, the emergence of antibiotic resistance in pathogenic bacteria is considered an important problem for society. Excessive consumption of antibiotics, long-term treatments, and inappropriate prescriptions continually increase the severity of the problem. Improving antibiotic stewardship requires improved diagnostic testing, and, therefore, in vitro antibiotic susceptibility testing is becoming increasingly important. This research details the development of an antibiotic susceptibility test for Mycobacterium smegmatis using streptomycin as antibiotics. This strain was selected because it is a member of the slow growing Mycobacterium genus and serves as a useful surrogate organism for M. tuberculosis. A commercially available and low-cost screen-printed gold electrode in combination with a specifically developed nucleic acid probe sequence for the 16SrRNA region of the mycobacterial genome was employed to monitor M. smegmatis nucleic acid sequences using the techniques of square-wave voltammetry and electrochemical impedance spectroscopy. The results show that it was possible to detect M. smegmatis sequences and distinguish antibiotic-treated cells from untreated cells with a label-free molecular detection. As a result, the in vitro antibiotic susceptibility test revealed that M. smegmatis showed sensitivity to streptomycin after a 24-H incubation, with the developed protocol representing a potential approach to determining antibiotic susceptibility more quickly and economically than current methods

POLİMERİK NANOPARTİKÜL FLORESAN PROBLARLA BAKTERİYAL FİLMLERİN İN-VİVO GÖRÜNTÜLENMESİ

The aim of this theses is to synthesize polymeric nanoparticle fluorescence probes in order to be used in in-vitro and in-vivo imaging of Staphylococcus aureus and the biofilms produced by these bacteria. 1ml THF solution containing certain amounts of PFBT “[poly(9,9-dioctylfluorene-2,7-diyl-co-benzothiadiazole)]” and DSPE-PEG2000-Maleimid were added to 9ml of DW. The ultra-sonication of this mixture was applied with 12W micro-tip probe sonication for 60s and steered overnight at the room temperature. At the end of this step, packaging of PFBT was done and CPDP-Mal nanocapsules (NCs) were synthetized successfully. Uv-vis spectrophotometery of the NCs was carried out and the results showed the characteristic peak shifting of PFBT from 470nm to 480nm that’s related to successful packaging of PFBT with DSPE-PEG2000-Mals. Zeta-sizer and TEM analysis showed that the prepared NCs have a diameter of 80nm in a narrow distribution. To gain specific targeting properties for obtained NCs, the immobilization of vancomycin (as a ligand) has been carried out in accordance with the protocol. For this purpose, bis-vancomycin molecules containing di-thiol bond were synthetized and Purification of bis-vancomycin was carried out by implementing HPLC and keeping it for three days at freeze-dryer. The LC-MS analysis of bis-vancomycin showed the related characteristic peaks. The di-thiol bonds of bis-vancomycin were cleaved using TCEP in aqueous environment and obtained SH-Van ligands have successfully immobilized onto CPDP-Mal NCs in accordance with the protocol, which was confirmed by the FTIR spectra of the ligands. Antibacterial properties of prepared SH-Van and CPDP-S-Van NCs were investigated by MIC, MBC and DDM. The in-vitro specific targeting properties of the CPDP-S-Van NCs were investigated on S. aureus and E. coli by confocal microscopy. Results showed that the NCs were targeted only to S. aureus. The NCs imaging abilities were examined on the biofilm which was produced in laboratory condition by S. aureus. The results manifested that the CPDP-S-Van NCs demonstrate high-level performance in the imaging of S. aureus biofilms. Cytotoxicity and apoptosis properties of NCs were investigated by MTT/WST-1 and caspase-3 antibody imaging assay. According to the results, cell viability was determined to be over 90% even at the highest dose and no apoptotic properties were detected for NCs. The results manifestly indicate that the prepared NCs can be used for the in-vivo studies. The ICR mouse animal model was used to inspect the specific imaging ability of CPDP-S-Van NCs against S. aureus in in-vivo conditions. Infected animal model was prepared by injecting S. aureus and E. coli bacteria subcutaneously into the right and left backside of the ICR mouse. Specific targeting abilities of NCs were investigated by using non-invasive live animal fluorescence imaging technique by the injection of CPDP-S-Van NCs from tail vein of infected animal model. According to the results the targeting of the prepared NCs has been done successfully.ÖZET I ABSTRACT III TEŞEKKÜRLER V SİMGELER VE KISATMALAR VI İÇİNDEKİLER VII ÇİZELGELER XI ŞEKİLLER XII 1. GİRİŞ 1 2. GENEL BİLGİLER 4 2.1. Nanoteknoloji 4 2.2. Nanomalzemeler 4 2.2.1. İnorganik Nanopartiküller 4 2.2.2. Organik Nanopartiküller 5 2.3. Organik Nanotaşıyıcılar 5 2.3.1. Nanokapsüller (Miseller, Lipozomlar) 6 2.3.1.1. Lipozomların Sınıflandırılması 6 2.3.1.2. İnkapsülasyon Teknikleri 8 2.3.1.3. Lipozom Sentezinde Kullanılan Lipitler 10 2.3.1.3.1. [1,2-Distearoyl-sn-glycerol-3-phosphatidylethanolamine–Poly(ethyleneglycol)] (DSPE-PEG) blok kopolimerler 12 2.3.2. Nanokapsüllerin Spesifik Olarak Hedeflendirilmesi 12 2.3.2.1. Biyolojik Hedeflendirme Ajanı (Ligand) 12 2.3.2.2. Antibiyotik Temelli Ligandlar 13 2.3.2.2.1. Vankomisin 15 2.4. Floresan Problar 17 2.4.1. Konjuge Polimerler (CP) 18 2.4.1.1. Poli(9,9-dioktilfloren- 2,7-diil-ko-benzotiadiazol) 20 2.5. Biyolojik Görüntülenme 21 2.5.1. Floresan Görüntülenme 23 2.5.2. Floresan Mikroskopu 23 2.6. Bakteri 25 2.6.1. Bakterilerin Hücresel Yapısı 25 2.6.2. Biyofilm 26 2.6.2.1. Biyofilm Yaşam Döngüsü 26 2.6.2.2. Biyofilmleri İçeren İnsan Bakteriyal Enfeksiyonları 27 2.7. Bakterilerin İn-vivo Floresan Görüntülenmesi 28 3. DENEYSEL ÇALIŞMALAR 30 3.1. Kullanılan Malzemeler 30 3.2. Floresan Probu Taşıyan DSPE-PEG2000 Malimid Fonksiyonelli Nanokapsüllerin Hazırlanması (CPDP-Mal NK’ler). 30 3.3. Tiyol Fonksiyonelli Vankomisin Hazırlanması 31 3.3.1. Hazırlanan Tiyol Uclu Vankomisinin Karakterizasyonu 33 3.3.2. Sentezlenmiş Bis-Vankomisin ve HS-Van’ların MALDI Kütle Spektroskopisi 33 3.4. Tiyol Uclu Vankomisinin CPDP-Mal NK’lerin Üzerine İmobilizasyonu 33 3.5. Elde Edilen NK’lerin Memeli Hücrelerinde Gösterdiği Sitotoksisite ve Apoptozis Etkilerinin Araştırılması. 35 3.5.1. WST-1 Tekniği ile CPDP-Mal NK’lerin L929 Fare Fibroblast Hücrelerin Üzerinde Gösterdiği Sitotoksisite Etkilerinin Araştırılması 35 3.5.2. Hazırlanmış CPDP-S-Van NK’lerin NIH/3T3 Fare Embriyonik Fibroblast Üzerinde Gösterdiği Sitotoksisite Özellikleri 36 3.5.3. Kazpas 3 Antikor Boyaması ile CPDP-Mal NK’lerin L929 Fare Fibroblast Hücrelerin Üzerinde Gösterdiği Apoptotik Etkilerinin Araştırılması 36 3.6. Hazırlanan HS-vankomisin, CPDP-S-Van NK’lerin Antibakteriyal Özelliklerinin Araştırılması 37 3.6.1. MİK ve MBK Çalışmaları 37 3.6.2. DDM Çalışmaları 39 3.7. Biyofilmlerin Üretimi 40 3.7.1. Üretilmiş Olan Biyofilm Niteliğinin Belirlenmesi 41 3.8. Biyofilm ile CPDP-S-Van NK’lerin Etkileşimi 42 3.9. Hazırlanan CPDP-S-Van NK’lerin Gram Pozitif Bakteriler İçin Hedeflendirme Özgüllüğünün Araştırılması 43 3.9.1. S. aureus Bakterisine Özel Hedeflendirilmiş CPDP-S-Van NK’lerin Konfokal Mikroskopu ile Belirlenmesi 44 3.9.2. CPDP-S-Van NK’lerin S. aureru Etkileşiminin Görüntülenmesi 44 3.10. İn-vivo Çalışmaları 45 3.10.1. İnfekte ICR Fare Modelinin Hazırlanması 45 3.10.2. CPDP-S-Van NK’lerin Farenin Enfekte Bölgesini Spesifik Olarak Görüntülenmesi 45 3.10.3. Hayvanın Canlı Floresan Görüntülenmesi 46 4. SONUÇ VE TARTIŞMA 47 4.1. CPDP-Mal NK’ler ve HS-Van Moleküllerin Karakterizasyonu 47 4.1.1. UV-Vis Spektrofotometre Analiz Sonuçları 47 4.1.2. Hazırlanmış HS-vankomisin’in Sıvı Kromatografisi-Gaz Spektroskopisi (LC-MS) ile Karakterizasyonu 48 4.1.3. Bis-Vankomisin ve HS-Van’ların MALDI Spektrometre Sonuçları 48 4.1.4. Hazırlanmış Bis-vankomisin ve CPDP-S-Van NK’lerin FTIR ile Karakterizasyon Sonucu 49 4.1.5. CPDP-Mal ve CPDP-S-Van NK’lerin DLS Analiz Sonuçları 51 4.1.6. CPDP-Mal ve CPDP-S-Van NK’lerin TEM Görüntülenme Sonuçları 52 4.2. Elde Edilen NK’lerin Memeli Hücrelerinde Gösterdiği Sitotoksisite ve Apoptozis Etkilerinin Araştırılması 53 4.2.1. CPDP-Mal NK’lerin WST-1 Yöntemi ile Elde Edilen Sitotoksisite Sonuçları 53 4.2.2. CPDP-S-Van NK’lerin 3 NIH/3T3 Fare Embriyonik Fibroblast Hücreleri Üzerinde Sitotoksisite Araştırma Sonucu 54 4.2.3. Kazpas 3 Antikor Boyama Tekniği Kullanarak CPDP-Mal NK’lerin Apoptoz Özelliklerin Belirlenmesi 55 4.3. Hazırlanan HS-vankomisin, CPDP-S-Van NK’lerin Antibakteriyal Özelliklerinin Araştırılması Sonucunda Elde Edilen Sonuçlar 57 4.3.1. MİK ve MBK Test Sonuçları 57 4.3.2. DDM Analiz Sonuçları 59 4.4. S. aureus (ATCC 25923), S. epidermidis (ATCC 12228) ve S. epidermidis (ATCC 35984) Bakteriler ile Hazırlanmış Biyofilmler 60 4.5. Biyofilm ile CPDP-S-Van NK’lerin Etkileşim Sonucu 63 4.6. CPDP-S-Van NK’lerin S. aureus’a Spesifik Bağlanabilme Özelliklerinin Araştırma Sonuçları 64 4.6.1. S. aureus Bakterisine Özel Hedeflendirilmiş CPDP-S-Van NK’lerin Konfokal Mikroskopu Ile Belirlenmesi 64 4.6.2. Hedeflendirilmiş CPDP-S-Van NK’lerin S. aureus ile Etkileşiminin TEM Görüntülenme Sonuçları 67 4.7. S. aureus Bakterisinin İn-Vivo Görüntülenmesi 69 5. GENEL SONUÇLAR 72 6. KAYNAKLAR 76 ÖZGEÇMİŞ 96Bu tezin amacı floresan özelliği gösteren konjüge polimer problarını kullanarak, gram pozitif olan Staphylococcus aureus bakterilerinin ve bu bakteri tarafından üretilmiş olan biyofilmlerin in-vitro ve in-vivo olarak görüntülenmesidir. Bu amaç için maleimid fonksiyonel grubuna sahip olan DSPE-PEG2000 kullanılmış, 60 saniye 12W güçünde olan mikro uçlu prob sonikatör ile 9 ml su ortamında 1 ml THF çözeltisi varlığında sonike edilmiş, gece boyunca oda sıcaklığında karıştırılmıştır. Böylece konjüge polimerlerin paketlenmesi gerçekleştirilmiş, CPDP-Mal nanokapsüllerin (NK) sentezi gerçekleştirilmiştir. Elde edilen NK’ler Uv-vis spektrofotometre ile karakterize edilmiş, PFBT’ye ait olan karakteristik pik 470 nm’den 480 nm’ye bir kayma göstermiştir. Oluşan bu pik kayması, PFBT probların DSPE-PEG2000-Mal tarafından paketlenme sonucunda ortaya çıkmıştır. Zeta-sizer ve TEM ile analiz sonucunda, bu NK’lerin dar bir boy dağılımı şeklinde 80nm çapına sahip oldukları tesbit edilmiştir. Hazırlanan NK’lere spesifik hedeflendirilme özelliği kazandırılması vankomisin ligandının immobilizasyonu ile gerçekleştirilmiştir. Bu amaç için vankomisin molekülleri SH grubu ile fonksiyonelleştirlmiştir. 100 mg satın alınmış vankomisin belli oranlarda DMSO, DMF, HBTU ve DIEA ile 0°C’da reaksiyona girerek di-tiyol bağına sahip bis-vankomisin elde edilmiş, HPLC ile saflaştırılarak üç gün kuru döndürücüde bekletilmiştir. LC-MS ile karakterizasyon sonuçları, bis-vankomisinin sentezlendiğini kanıtlamıştır. TCEP kullanarak di-tiyol bağları koparılmış, elde edilen HS-Van molekülleri sulu bir ortamda CPDP-Mal NK’lerine immobilizasyonu gerçekleştirilmiştir. FTIR analizi ile alınan sonuçlar, vankomisin moleküllerin başarılıyla NK’lere bağlandığını göstermiştir. Sentezlenen HS-Van ve CPDP-S-Van Nk’lerin antibakteriyal özellikleri MİK, MBK ve DDM ile araştırılmıştır. Hazırlanmış olan CPDP-S-Van NK’lerin spesifik hedeflendirilme özellikleri in-vitro koşullarında S. aureus ve E. coli üzerinde araştırılmış, bu NK’lerin başarılı bir şekilde sadece gram pozitif olan S. aureus’a bağlanması görüntülenmiş/belirlenmiştir. Lab koşullarında S. aureus tarafından üretilmiş biyofilmlerin görüntülenebilmesini araştırmak için hazırlanmış CPDP-S-Van NK’ler kullanılmıştır, biyofilmlerin görüntülenmesini gerçekleştirmiştir. NK’lerin sitotoksisite özellikleri MTT testi ile araştırılmış, elde edilen sonuçlara göre kullanılabilecek en yüksek dozda dahi, %90 üzeri hücre canlılığı tesbit edilmiştir. Apoptoz ve nekroz testlerinden alınan sonuçlar ise hücrelerde her hangi bir toksik etki göstermemiştir. Elde edilen sonuçlar, hazırlanmış NK’lerin in-vivo ortamında kullanılabileceğinin bir güvencesidir. CPDP-S-Van NK’lerin in-vivo olarak S. aureus’u spesifik olarak görüntülebilmesini araştırmak için ICR fare hayvan modeli kullanılmıştr. S. aureus ve E. coli bakterileri subkütan olarak fare sırtının sağ ve sol bölgelerine enjekte edilerek, infekte hayvan modeli elde edilmiştir. İnfekte hayvan modeline kuyruk damarından verilmiş NK’lerin hedeflendirilebilme özellikleri non-invazif canlı hayvan floresan görüntülenme tekniğini kullanarak Maestro EX floresan sistem cihazı ile araştırılmıştır. Sonuçlara göre hazırlanmış olan NK’lerin hedeflendirilmesi başarılıyla gerçekleştirilmiştir

In vivo imaging/detection of MRSA bacterial infections in mice using fluorescence labelled polymeric nanoparticles carrying vancomycin as the targeting agent

Norouz Dizaji, Araz/0000-0001-6720-2115WOS: 000498377700001PubMed: 31762403This study aims to develop fluorescence labelled polymeric nanoparticle (NP) carrying vancomycin as the targeting agent for in vivo imaging of Methicillin-resistant Staphylococcus aureus bacterial infections in animal models. Maleimide functionalized 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000] as the main was carrier matrix to prepare the NPs. A fluorescence probe, namely, poly[9,9 '-bis (6 ''-N,N,N-trimethylammonium) hexyl) fluorene-co-alt-4,7-(2,1,3-benzothiadiazole) dibromide] was encapsulated within these NPs by ultrasonication successfully. UV-Vis spectro- photometry of the NPs showed the characteristic shifting on the peak of conjugated polymers indicating successful packaging of this compound with lipid bilayers in nanoscales. Zeta-sizer and TEM analysis showed that the prepared NPs have a diameter of 80-100 nm in a narrow size distribution. Thiolated vancomycin was synthesized and attached to the NPs as the targeting agent. FTIR and MALDI-TOF spectroscopy analysis confirmed the immobilization. The specific targeting properties of the vancomycin conjugated NPs to the target bacteria were first confirmed in in vitro bacterial cultures in which Escherichia coli was the non-target bacteria - using confocal microscopy and TEM. Imaging of bacterial infections in vivo was investigated in mice model using a non-invasive live animal fluorescence imaging technique. The results confirmed that bacterial infections can be detected using these novel polymeric NPs carrying fluorescence probes for imaging and vancomycin as the targeting agent - in vivo successfully.Turkish Scientific and Technological CouncilTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [1130864]This study was conducted in the frame of a EU project between Europe and China - the shortly called 'ABREM', 'FP7-PEOPLE-2009-IRSES' and also supported by Turkish Scientific and Technological Council (Project Number: 1130864)

Highly sensitive label-free electrochemical detection of heat shock protein with low-cost screen-printed electrodes

Heat shock proteins (HSPs) are produced when organisms are exposed to various environmental stress conditions such as extreme temperatures, light, and toxins. It is a known fact that in bacteria which have the HSP gene (hsp), antibiotics can trigger the expression of these proteins. However, the response of HSP genes to antibiotics has not been fully clarified in the literature, with studies still ongoing. In this work, a novel method of detecting heat shock protein 65 (HSP65) was investigated using electrochemical impedance spectroscopy (EIS) due to its sensitivity and selectivity. To do so, a specific HSP65 probe and target were designed and their hybridization behaviour was studied using low-cost screen-printed electrodes (SPEs). Cyclic voltammetry was performed to analyse surface characteristics of the SPEs and the performance of the electrodes was tested using EIS, by measuring changes in the charge transfer resistance upon probe binding and target hybridization. Increases in charge transfer resistance were measured and observed to be in-line with literature. Based on these results, the designed HSP65 probe was confirmed to bind the target sequence and proved that the EIS can be effectively used to detect HSP65 label-free. Results presented here could lead to development of antibiotic susceptibility assays based on hsp genes in future and provide a quicker test for the detection of many slow-growing bacteria

An electrochemical biosensor with integrated microheater to improve the sensitivity of electrochemical nucleic acid biosensors

Electrochemical impedance spectroscopy (EIS) is often used for biomolecular detection based on the interaction of a molecule with a receptor functionalised electrode surface and consequent impedance change. Though its performance is well established, there is still a need for improved sensitivity and specificity, especially when attempting to detect nucleic acids from clinical samples with minimal amplification steps. Localised heating is a potential approach for improving nucleic hybridisation rates and reducing non-specific interactions, and thereby producing high sensitivity and selectivity [1-3]. The aim of the study was therefore to develop a microheater surrounding Au thin film electrodes, an integrated hybrid chip, for detecting genes of Mycobacterium tuberculosis with enhanced sensitivity. The performance of the integrated hybrid chip was determined using the changes in the charge transfer resistance (Rct) upon DNA hybridisation using probe sequences for Mycobacterium tuberculosis. Heat transfer within the system was simulated by using COMSOL Multiphysics as a mathematical modelling tool. When a temperature of 50 °C was applied to the microheater during DNA hybridisation steps, Rct values (which were indicative of DNA-DNA hybridisation) increased 236% and 90% as opposed to off-chip non-heated experiments and off-chip heated experiments. It is concluded from these observations that the microheater indeed can significantly improve the performance of the nucleic acid hybridisation assay and paves the way for the development of highly- sensitive and specific integrated label-free biosensors

Simple and low cost antibiotic susceptibility testing for mycobacterium tuberculosis using screen-printed electrodes

One quarter of the global population is thought to be latently infected by Mycobacterium tuberculosis (TB) with it estimated that 1 in 10 of those people will go on to develop active disease. Due to the fact that Mycobacterium tuberculosis (TB) is a disease most often associated with low and middle income countries it is critical that low cost and easy to use technological solutions are developed which can have a direct impact on diagnosis and prescribing practice for TB. One area where intervention could be particularly useful is antibiotic susceptibility testing (AST). In this work presents a low cost, simple to use AST sensor which can detect drug susceptibility on the basis of changing RNA abundance for the typically slow growing Mycobacterium tuberculosis (TB) pathogen in 96 hours using screen-printed electrodes and standard molecular biology laboratory reactionware. In order to find out sensitivity of applied sensor platform, different concentration (108-103 CFU/mL) of M. tuberculosis was performed and Limit of detection (LOD) and limit of quantitation (LOQ) was calculated as 103.82 and 1011.59 CFU/mL, respectively. The results display that it was possible to detect TB sequences and distinguish antibiotic-treated cells from untreated cells with a label-free molecular detection. These findings pave the way for development of a comprehensive, low cost and simple to use AST system for prescribing in TB and mdrTB

A fully integrated rapid on-chip antibiotic susceptibility test - a case study for Mycobacterium smegmatis

Antibiotic resistance is one of the most pressing scientific and societal issues of our age. There is an urgent need to develop new diagnostic technologies which can quickly determine whether an infection is susceptible or resistant to different treatments so that rational antibiotic prescribing can take place. The main objective of the study was therefore to develop a rapid, simple, cost effective and comprehensive antibiotic susceptibility/resistance test based on rapid nucleic acid profiling. To do so, we integrated a microelectrode sensor within a microfluidic chip that combined bacterial incubation, lysis, and electrochemical detection chambers in a single simple set-up. As a case study, Mycobacterium smegmatis was investigated as a surrogate organism for Mycobacterium tuberculosis. The novelty of the work lies in developed capability of performing incubation, lysis, fragmentation, and detection process in a comprehensive yet simple lab-on-a-chip device called ‘MycoCHIP’. A gold microelectrode in combination with a specifically developed nucleic acid probe sequence for the 16SrRNA region of the mycobacterial genome were employed to monitor M. smegmatis nucleic acid sequences using Differential Pulse Voltammetry (DPV) and Square-Wave Voltammetry (SWV). The results demonstrated that it was possible to detect bacterial nucleic acid sequences and distinguish antibiotic incubated (Ab-i) cells from nonincubated (Ab-n) cells on MycoCHIP with a label-free molecular detection. The antibiotic susceptibility test showed that through measuring 16SrRNA levels from M. smegmatis, sensitivity to antibiotic was apparent after 24 h incubation, with a developed protocol representing a potential approach to determining antibiotic susceptibility more quickly, reliable and economically than current methods