Haider Sabah Abdülhüsein Thiaqr Üniversite Biyoloji bölümü Biyosenserler Biyosensörler • Biyosensörlerin tarihi 50’li yılların ortalarında L.C.Clark’ın (Ohio,ABD) Cincinnati ameliyat sırasında Hastanesi’nde kanın O2 miktarını bir elektrod ile izlemesiyle başlar.1962 yılında Clark ve Lyons Glukozoksidaz(GOD) enzimini O2 elektrodu ile kombine ederek kanın glukoz düzeyini ölçmeyi başardılar. Biyosensör Terimi 1- Analyte: a sample that have cell or another materiles ( Ab, Ag, enzyme , amıno acid ete.) 2- Bioreceptor: Molecular recognizing materials 3- Transducer: signal transducer 4- Electric signal ( measurable signal) 5- Detector Biyosensörler (biyoalgılayıcılar), bünyesinde duyargacı biyolojik bulunan olarak ve bir çeviriciyle fizikokimyasal birleştirilmiş bir analitik cihazlar tanımlanmaktadır. Bir biyosensörün amacı, bir veya bir grup analiz edilecek madde miktarıyla orantılı olarak sürekli sayısal elektrik sinyali üretmektir. Tipik Biyosensörlerin bileşenleri Operating Principles Biosensors are constituted by three components. These devices have sensing elements, also called bioreceptor that emulates in vivo molecular recognition phenomena. There is a wide range of sensing elements such as cells, microbes, cell receptors, antibodies, enzymes, or nucleic acids. These biological sensitive elements recognize the analyte and interact with it depending on the type of biosensor. One of the main biorecognition strategies is based on bacterial or viral nucleic acid sequences. Biyosensörlerin, en önemli ikinci kısmı da “Çevirici (Transducer)” bölümüdür. Çeviriciler, biyoajan-analit etkileşmesi sonucu gerçekleşen fizikokimyasal sinyali elektrik sinyaline dönüştürerek, bu sinyalin daha sonralari güçlenerek okunabilir ve kaydedilebilir bir şekle girmesine öncülük ederler. The last part of a biosensor is the reader device. It usually involves a display that depends on software and hardware to generate the results Biyosensör operating system Neden Biyosensörler kullanıyoruz ? • Virus and bacteria detection commonly involves the use of several molecular techniques such as the reverse transcription-polymerase chain reaction (RTPCR), which remains the gold standard for pathogen detection. The classical detection methods for these pathogens usually require isolation, culturing and, biochemical tests. Additionally, serological tests like the (ELISA) are used for the detection of antibodies and immunoglobulin needed for identification purposes. However, some of these techniques take a long time to obtain results. Biyosensörler türleri Biosensors can be classified by the way they transduce signals into optical, electrochemical, and piezoelectric devices : 1- Optical biosensors are those that perform their analysis through the measure of photons, using optic fibers as transduction elements. Several optic sensing mechanisms can be employed by this type of biosensor for analyte detection such as absorption, colorimetry, fluorescence, or luminescence . This kind of biosensor presents a lower noise and immunity to electromagnetic interference. Optical geometry of biosensor Surface Plasmon Resonance (SPR) is an optical technique that has contributed greatly to immunoassays development 2- Electrochemical biosensor: has been extensively applied to pathogen detection. A chemical sensor is a device that transforms chemical information, such as the concentration of a specific sample component or total compositional analysis into an analytically useful signal. 3- Piezoelectricity refers to the ability of a material to generate a voltage under mechanical stress. These biosensors possess crystals that vibrate under the influence of an electric field. Besides, certain materials vibrate at characteristic resonant frequencies in response to interaction with other molecules. BİYOSENSÖR ELEKTROKİMYASAL BİYOSENSÖR Ampero metrik Bazlı Voltmetrik Bazlı Potansiyo metrik Bazlı KÜTLE BAZLI BİYOSENSÖR OPTİK BİYOSENSÖR Biyolüminisans Bazlı İmpedim etrik Bazlı Kolorimet rik Bazlı Manyetoelektri k Bazlı Piezoelektr ik Bazlı Floresans Bazlı Kondüktometr ik Bazlı BİYOSENSÖRLERİN SINIFLANDIRILMASI KALORİMETRİ K BİYOSENSÖR 1- Transduction Electrochemical biosensor is an electrochemical cell where the main component is commonly a working electrode. Electrodes can be fabricated from multiple materials and using various manufacturing processes. An electrode is an electronic conductor through which charge is transported by the movement of electrons and holes. Electrodes are thus fabricated from conducting and semiconducting materials, including metals, such as gold (Au), and nonmetals, such as carbon. Manufacturing processes can be used to fabricate electrodes of various sizes, including bulk structures (greater than 1 mm) and micro- and nano-structures. Electrodes can be classified by type and form of material, manufacturing process, and design. Electrode designs can be classified by form factor • Planar, wire, nanostructured, or array-based. The material, fabrication approach, and design affect the electrode’s structure and properties, which ultimately determine the biosensor’s performance, including sensitivity, selectivity, limit of detection (LOD), and dynamic range. 1-Metal electrodes Such as Au and platinum (Pt), have been commonly used for detection. pathogen Thick metal electrodes are commonly fabricated from structures via processes bulk cutting 2-Ceramic electrodes Conducting and semiconducting ceramics, including indium tin oxide (ITO), polysilicon, and titanium dioxide (TiO2) have also been examined for pathogen detection. For example, a silicon electrode for Salmonella typhimurium detection 3-Polymer electrodes Polymers have various advantages, including tunable electrical conductivity, biocompatiblity, and environmentally stability. Polymer electrodes can be broadly classified as (1) conjugated polymer or (2) polymer composite. 2- Biorecognition elements 1. Antibodies and antibody fragments are among the most commonly utilized biorecognition elements for pathogen detection using electrochemical biosensors. Biosensors employing antibody- based biorecognition elements are commonly referred immunosensors. to as 2-Carbohydrate-binding proteins Such as lectins, also provide selective biorecognition elements for pathogen detection based on their ability to selectively bind ligands on target species. Peptidebased biorecognition elements are relatively low-cost, produced with automated synthesis and are modifiable. can high be yield processes, 3-Oligosaccharides Trisaccharides are carbohydrates that can selectively bind carbohydrate-specific receptors on pathogens. ligands Thus, have biorecognition pathogen trisaccharide been used elements detection electrochemical biosensors. as for using 4-Oligonucleotides ssDNA is commonly used as a biorecognition element for DNA-based assays, ssDNA aptamers are commonly used for pathogen detection using electrochemical biosensors. Aptamers are single-stranded oligonucleotides capable of binding various molecules with high affinity and selectivity. Aptamers are isolated from a large random sequence pool through a selection process that utilizes systematic evolution of ligands by exponential enrichment, also known as SELEX. Aptamer-based detection of influenza viruses. Schematic representations of aptamer development and virus detection. Selex procedure is applied for selection of specific aptamers. These sensing elements are immobilized on the sensor surface to bind efficiently to the viral proteins in infected samples. The recognition signal is proceeded to provide diagnostic. • SELEX high affinities for C. parvum for detection in fruit samples. However, the use of aptamers as biorecognition elements has not yet replaced traditional biorecognition elements, such as antibodies, because of several challenges, such as aptamer stability, degradation, cross-reactivity, and reproducibility approaches. using alternative processing 5. Phages Are viruses that infect and replicate in bacteria through selective binding via tail-spike proteins Thus, they have been examined as biorecognition elements for pathogen detection using electrochemical biosensors. Biyosensörler, genel olarak analizlenecek madde ile seçimli bir şekilde etkileşime giren biyoaktif bir bileşenin, bu etkileşim sonucu ortaya çıkan sinyali ileten bir iletici sistemle birleştirilmesi ve bunların bir ölçüm sistemiyle kombinasyonuyla oluşturulurlar. Biyosensörler için mümkün uygulama alanları şunlardir: • Klinik diyagnostik, biyomedikal sektör • Proses kontrolü: Biyoreaktor kontrol Gıda uretim ve analızı • Tarla tarımı, bag-bahçe tarımı ve veterinerlik • Bakteriyel ve viral diyagnostik • Endüstriyel atık su kontrolu • Çevre koruma ve kirlilik kontrolu • Madden ve işletmelerinde toksik gaz analizleri • Askeri uygulamalar Biyosensörlerin uygulamaları Sensörler türleri • Analit-Biyoaktif madde ilişkisine göre Biyosensörlerin sınıflandırılması: • 1-Biyoaffinite esaslı biyosensörler • 2-Biyokatalitik esaslı biyosensörler • 3- İmmobilize hücre esaslı biyosensörler • 4-Transmembran esaslı Biyosensörler Biyoaffinite Esaslı Biyosensörler: (örneğin; iletici sistem üzerinde antikor) immobilizasyonuyla antijenlerin tayini) *Biyokatalitik Esaslı Biyosensörler (örneğin; iletici sistem üzerinde enzim immobilizasyonuyla enzimin substratı, inhibitörü, aktivatörü veya koenzimi olan çeşitli kimyasal maddelerin tayini). *İmmobilize Hücre Esaslı Biyosensörler (örneğin; iletici sistem immobilizasyonuyla o üzerinde hücrelerin hücreler tarafından metabolize edilen çeşitli maddelerin tayini) Transmembran Esaslı Biyosensörler (örneğin; çeşitli moleküllere spesifik reseptör veya farklı membran proteinlerini içeren hücre membranlarının iletici sistem üzerinde immobilizasyonuyla söz konusu moleküllerin seçimli bir şekilde tayinleri.) Reseptör Tutuklanması (immobilizasyonu) • Biyoaktif bileşen ile iletici unsurun birleştirilmesinde oldukça farklı immobilizasyon yöntemlerinden yararlanılabilir. Biyoaktif bileşen sensör olarak da adlandırabileceğimiz temel iletici unsur üzerinde fiziksel olarak, jel içinde veya polimer matrikste tutuklanabilir, kovalent edilebilir. veya elektrot çapraz yüzeyinde bağlanarak biriktirilebilir, immobilize İmmobilizasyonu Patojen tespiti için elektrokimyasal biyosensörler kullanmaktadır Conventional methods for viral detection include virus or microorganism propagation and isolation from culture. These methods are effective and sensitive but tend to be costly, labor intensive and time consuming (typically results are available in 2–10 days). Alternative molecular methods based on polymerase chain reaction (PCR), real time PCR (RT-PCR) are more specific, sensitive and take less time, but they need isolated genetic materials, manipulation with special care and necessitate sophisticated equipment, and, thus, they are hardly to be applied for on-site monitoring. Consequently, development of a valid diagnostic assay for swift pathogen detection and identification, with high sensitivity and selectivity is a challenge for researchers all over the world. In the first step of detection a specific influenza virus biomarker is recognized by antibodies carried by gold-nanoparticles pre-adsorbed on the conjugate pad. Usually antibodies raised against preserved epitopes in viral nucleoprotein are used for this step. Influenza viruses complexed with immune-gold nanoparticles reach the test lines. Two test lines with pre-immobilized antibodies that specifically recognize either influenza A or B virus bind to different epitopes of the virus and, in that way accumulate immune-gold nanoparticles carrying the viruses. The accumulation of gold nanoparticles results in an appearance of a visible red line. Usually, antibodies recognizing specific epitopes in HA proteins are used for the test line. Finally, non-bound immune-gold nanoparticles arrive at the control line which harbors the secondary antibody, showing the second visible red line. In the absence of viral particles in the sample, the immune-gold nanoparticles flow alone and bind only to the control line. Thus, two colored lines stand for positive result while a single colored line corresponds to negative result Advanced biosensors for detection of pathogens related to livestock and poultry Jasmina Vidic1*, Marisa Manzano2, Chung-Ming Chang3 and Nicole Jaffrezic-Renault4 an electrochemical immunosensor based on a specific anti-M1 antibody was shown to detect all serotypes of influenza A virus with sensitivity similar to classical molecular methods (80–100 × 103 PFU/mL). A lower effective limit of 1 × 103 PFU/mL was achieved by coupling the anti-M1 monoclonal antibody to gold nanoparticles in a quartz crystal microbalance assay A sensitive plasmon-assisted fluoro-immunoassay was developed for the detection of the influenza virus by specific anti-M1 antibodies conjugated to gold nanoparticle-decorated carbon nanotubes. After influenza virus binding to these mixed nanoparticles, a fluorescent signal was produced by addition of cadmium telluride quantum dots. A photoluminescence intensity of quantum dots was shown to vary as a function of virus concentration, with a detection limit of 50 PFU/mL. • Cell-based biosensors have also contributed to COVID-19 diagnosis. Mavrikou et al. developed a biosensor based on membrane-engineered mammalian cells that possess the human chimeric spike. S1 antibody. The device can detect SARS-CoV-2 S1 spike protein selectively, where the binding of the protein to the membrane-bound antibodies results in cellular bioelectric properties modification measured by Bioelectric Recognition Assay. Figure 4. COVID-19 rapid serological IgM/IgG test. Reprinted with permission from Ghaffari, A. Et al. COVID-19 Serological Test: How Well Do They Actually Perform? Diagnostics 10(7): 453. Copyright (2020) MDPI Sample preparation: • Including concentrating or amplifying the target species through separation and growth processes, reducing the concentration of background inhibitory species, and reducing the heterogeneity of composition and properties the sample’s 1. Sample filtration • For example, biorecognition elements that exhibit affinity to a broad group of pathogens, such as lectins, have been used in pre-concentration steps for pathogen detection 2. Centrifugal separation • Centrifugation can be used as a density gradient-based concentrating sample. separation target principle pathogens Centrifugation-based within for a separation techniques can also potentially be applied to microfluidic-based biosensing platforms. 3-Broth enrichment • Is a technique used to increase the concentration of target species in the sample through growth or replication of target species prior to measurement, thereby increasing the number present for detection. The technique is commonly used in food safety applications. 4. Magnetic separation • The separation of the target species from a sample using magnetic beads has become a preparation commonly used approach in detection applications. sample pathogen Electrochemical methods for pathogen detection using electrochemical biosensors • 1. Potentiometric methods, also referred to as controlled-current methods, are those in which an electrical potential is measured in response to an applied current . An advantage of controlledcurrent methods is the ability to use low-cost measurement instrumentation relative to that required for controlled-potential methods. 2-Voltammetric methods • Also referred to as controlled-potential methods, are those in which a current is measured in response to an applied electrical potential that drives redox reactions.