Name: phthalic acid ester-binding dna aptamer selection, characterization, and application to an electrochemical aptasensor
Uploaded: 2018-03-21T12:30:00.000+00:00
Duration: 9 min 33 s
Description: Watch this Scientific Journal Video about Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor at JoVE.com

ونحن ممتنون للدعم المالي من "مؤسسة العلوم الطبيعية الوطنية" (21675112)، المشاريع الرئيسية لخطة لجنة التعليم ببكين (KZ201710028027) ويانجينغ الشباب الباحث البرنامج من رأس المال الطبيعي جامعة العلوم والتكنولوجيا.

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الكتاب يعلن أي مصلحة مالية المتنافسة.

فائدة المعلقة واحد من الابتامرات أن تحديدها من خلال الأسلوب في المختبر سيليكس، بينما الأجسام المضادة يتم إنشاؤها عن طريق إيمونوريكشنز في فيفو . ولذلك، يمكن تحديد الابتامرات مع الهدف المنشود خصوصية ظروف تجريبية مصممة تصميماً جيدا، بينما الأجسام المضادة تقتصر على الظروف الفسيول...

جنبا إلى جنب مع التنمية الاقتصادية السريعة، وتسريع التصنيع والبناء الحضري والتلوث البيئي أكثر حدة من أي وقت مضى. وتشمل الملوثات البيئية النموذجية أيونات المعادن الثقيلة والسموم والمضادات الحيوية، ومبيدات الآفات، واختلال الغدد الصماء والملوثات العضوية الثابتة (POPs). أيونات المعادن والسموم، وإلى جانب الملوثات الأخرى جزيئات صغيرة جداً التي غالباً ما تتألف من مجموعة متنوعة من متجانسات. على سبيل المثال، تتضمن الملوثات العضوية الثابتة الأكثر سمية ثنائي الفينيل متعدد الكلور (PCBs)، والهيدروكربونات العطرية (العطرية)، ثنائيات الفينيل متعدد الكلور الاثير ثنائي الفينيل، وثنائي بنزو-فالديوكسينات (PCDDs)، ديبينزوفوران متعدد الكلور (PCDFs)، والافثاليك استرات حمض (آيس)1،2، التي تتكون من العديد من متجانسات كافة. وقد أجريت الكشف عن جزيء صغير أساسا بالأساليب المستندة إلى الطيف اللوني/الجماهيري نظراً لتنوعها من التطبيقات3،4،،من56. المكتشفة في الموقع، كانت الأساليب المستندة إلى جسم مؤخرا نمواً7،،من89. ومع ذلك، نظراً لهذه الأساليب محددة للغاية لمتجانسات معينة، يجب إجراء اختبارات متعددة. ما أخطر أن المتجانسات الرواية التي تنمو بسرعة أنه لا يمكن إنشاء تلك الأجسام المضادة في الوقت المناسب. ولذلك، وضع أجهزة الاستشعار الخاصة بفريق لرصد المستويات الإجمالية لجميع المتجانسات في اختبار واحد قد توفر مقياس لا تقدر بثمن لتقييم حالة التلوث البيئي.
في الآونة الأخيرة، الابتامرات الحمض النووي طبقت على نطاق واسع كعناصر الاعتراف في مختلف المنابر بيوسينسينج بسبب قدرتها على الاعتراف بطائفة واسعة من الأهداف، من الأيونات والجزيئات الصغيرة للبروتينات والخلايا10،11 ،12. يتم تحديد الابتامرات من خلال أسلوب في المختبر يسمى التطور المنهجي من يغاندس بتخصيب الأسية (سيليكس)13،14. ويبدأ سيليكس المكتبة اليغنوكليوتيد عشوائي جديلة واحدة الاصطناعية، والذي يحتوي على حوالي 1014-1015 تسلسل. حجم المكتبة عشوائي يضمن تنوع هياكل مرشح الجيش الملكي النيبالي أو الحمض النووي. عملية سيليكس نموذجي يتكون من جولات متعددة لتخصيب اليورانيوم حتى هو إثراء المكتبة في تسلسل مع تقارب عالية وخصوصية إلى الهدف. ثم يتم تعيين تسلسل تجمع التخصيب النهائي، وثوابت التفكك (كد) وانتقائية ضد إمكانية التدخل المواد تتحدد بواسطة تقنيات مختلفة مثل تصفية ملزمة، تقارب اللوني، السطحية صدى مأكل مثل الطحين (موارد البرنامج الخاصة)، إلخ. 15
نظراً لقابلية الذوبان في الماء سيئة للغاية وعدم وجود المجموعات الوظيفية لتجميد السطحية، التحديد ابتمر من الملوثات العضوية الثابتة من الصعب نظرياً. وقد تقدما ملحوظا سيليكس الإسراع باكتشاف الابتامرات. ومع ذلك، اختيار مجموعة محددة الابتامرات للملوثات العضوية الثابتة لم قد يبلغ بعد. حتى الآن، كانت فقط الدنا ثنائي الفينيل متعدد الكلور-ربط الابتامرات مع خصوصية عالية لمتجانسات معينة حددت16. آيس تستخدم أساسا في مواد البولي فينيل كلوريد، تغيير البولي فينيل كلوريد من بلاستيك الصلب البلاستيك مرنة، وبالتالي بوصفها الملدنات. بعض آيس قد حددت كعوامل اختلال الغدد الصماء، ويمكن أن تسبب ضررا خطيرا للكبد ووظائف الكلي، تقليل حركية الحيوانات المنوية الذكور، وقد تسفر عن مورفولوجيا الحيوانات المنوية غير طبيعي وسرطان الخصية17. وأبلغ مجمع-وﻻ الابتامرات PAE الملزمة الخاصة بالمجموعة.
والهدف من هذا العمل تقديم بروتوكول ممثل لاختيار مجموعة محددة الحمض النووي الابتامرات إلى جزيئات صغيرة جداً مسعور مثل آيس، مجموعة تمثيلية من الملوثات العضوية الثابتة. ونحن أيضا بيان تطبيق ابتمر المحددة للكشف عن التلوث البيئي. يوفر هذا البروتوكول توجيهات ورؤى لاكتشاف ابتمر الجزيئات الصغيرة الأخرى مسعور.

<table><tbody><tr><td>UV-2550</td><td>Shimadzu,Japan</td><td></td><td>protocol, section 3.8.2</td></tr><tr><td>DNA Engnine Thermal cycler,PTC0200</td><td>BIO-RAD</td><td></td><td>section 3.5.1.2 and 3.5.2</td></tr><tr><td>C1000 Touch</td><td>BIO-RAD</td><td></td><td>section 5.3.6 and 6.3</td></tr><tr><td>VMP3 multichannel potentiostat</td><td>Bio-Logic Science, Claix, France</td><td></td><td>section 7.4,7.8 and 7.11</td></tr><tr><td>Epoxy-activated Sepharose 6B</td><td>GE Healthcare (Piscataway, NJ, USA)</td><td>10220020</td><td>argarose beads, section 2.3 and 3.3</td></tr><tr><td>Dynabeads M-270 carboxylic acid magnetic beads</td><td>Invitrogen, USA</td><td>420420</td><td>magnetic beads,section 5.2. and 5.3</td></tr><tr><td>Premix Taq Hot Start Version</td><td>Takara,Dalian,China</td><td>R028A</td><td>polymerase, section 3.5.1.1</td></tr><tr><td>PARAFILM Sealing Membrane</td><td>Bemis, USA</td><td>PM-996</td><td>section 3.6.5</td></tr><tr><td>Lambda Exonuclease</td><td>Invitrogen, USA</td><td>EN0561</td><td>section3.7.1.2.The 10 &amp;times; reaction buffer is provided along with &amp;lambda; exonuclease by the provider.</td></tr><tr><td>Dr. GenTLE&lt;br/&gt; Precipitation Carrier</td><td>Takara,Dalian,China</td><td>9094</td><td>section 3.6.2 and 3.8.1</td></tr><tr><td>UNIQ-10 PAGE DNA recovery kit</td><td>Sangon Biotech (Shanghai)</td><td>B511135</td><td>section 4.2</td></tr><tr><td>SYBR Gold nucleic acid gel stain</td><td>Invitrogen, USA</td><td>1811838</td><td>nucelic acid stain dye, section 3.5.1.5</td></tr><tr><td>SYBR Premix Ex Taq II</td><td>Takara,Dalian,China</td><td>RR820A</td><td>polymerase mix contaning polymerase and dNTPs, section 5.3.5</td></tr><tr><td>2-(N-Morpholino)ethanesulfonic acid (MES)</td><td>Sigma-Aldrich</td><td>CAS: 1132-61-2</td><td>section 5.2.1</td></tr><tr><td>1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC)</td><td>Invitrogen, USA</td><td>CAS: 25952-53-8</td><td>section 5.2.2</td></tr><tr><td>N-hydroxysuccinimide (NHS)</td><td>Sigma-Aldrich</td><td>6066-82-6</td><td>section 5.2.3</td></tr><tr><td>mercaptohexanol (MCH)</td><td>Sigma-Aldrich</td><td>CAS: 1633-78-9</td><td>section 7.7</td></tr><tr><td>Gold electrode</td><td>Shanghai Chenhua</td><td>CHI101</td><td>section 7.4. - 7.11</td></tr><tr><td>tris(2-carboxyethyl) phosphine hydrochloride (TCEP)</td><td>Sigma-Aldrich</td><td>CAS: 51805-45-9</td><td>section 7.5</td></tr><tr><td>O-(2-Mercaptoethyl)-O&#039;-methyl-hexa-(ethylene glycol)</td><td>Sigma-Aldrich</td><td>CAS: 651042-82-9</td><td>section 7.7</td></tr><tr><td>diethylhexyl phthalate (DEHP)</td><td>National Institute of Metrology, China</td><td>CAS: 117-81-7</td><td>section 7.11</td></tr><tr><td>Tween 20</td><td>Sigma-Aldrich</td><td>CAS: 9005-64-5</td><td>polyoxyethy-lene(20) sorbaitan monolaurate</td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>CAS: 9002-93-1</td><td>non-ionic surface active agent</td></tr><tr><td>PBS</td><td>Sigma-Aldrich</td><td>P5368</td><td>10 mM phosphate buffer containing 1 M NaCl, pH 7.4</td></tr></tbody></table>

phthalic acid ester-binding dna aptamer selection, characterization, and application to an electrochemical aptasensor

حمض فثاليك أرون استرات (آيس) من المجموعات الرئيسية للملوثات العضوية الثابتة. الكشف عن مجموعة محددة من آيس المطلوب هو درجة عالية نظراً لتزايد سريع للمتجانسات. الابتامرات الحمض النووي قد طبقت متزايدة كعناصر الاعتراف على منصات بيوسينسور، ولكن الابتامرات تحديد اتجاه أهداف جزيء صغير جداً مسعور، مثل آيس، نادراً ما يقال. هذا العمل وصف أسلوب المستندة إلى حبة مصممة لتحديد الابتامرات الحمض النووي الخاصة بالمجموعة لايس. المجموعة الأمينية فونكتيوناليزيد والفثالات الفثالات (دبة-NH2) المستهدف مرساة توليفها ومعطلة في الخرز [اغروس] تنشيط الإيبوكسي، مما يتيح عرض مجموعة إستر الافثاليك في سطح المصفوفة التثبيت، و ولذلك اختيار الاضبارات الخاصة بالمجموعة. حددنا الثوابت تفارق المرشحين ابتمر بتفاعل سلسلة البلمرة الكمية مقترنة بالفصل المغناطيسي. الوشائج النسبية والانتقائية في الابتامرات لايس أخرى تحددها الاختبارات التنافسية، حيث كان يحدها المرشحين ابتمر مسبقاً إلى دبة-NH2 تعلق الخرز المغناطيسي وصدر للمادة طافية على الحضانة مع آيس تم اختبارها أو مواد أخرى التدخل المحتملة. تم تطبيق التحليل التنافسي لأنها تتيح مقارنة السطحية تقارب بين آيس أن كان لا من المجموعات الوظيفية للتثبيت السطحي. أخيرا، أظهرت تلفيق أبتاسينسور الكهروكيميائية وتستخدم للكشف عن bis(2-ethylhexyl) فتالات أولتراسينسيتيفي وانتقائية. يوفر هذا البروتوكول رؤى لاكتشاف ابتمر الجزيئات الصغيرة الأخرى مسعور.

نحن تصميم وتوليفها فثالات والفثالات المجموعة الأمينية فونكتيوناليزيد (دبة-NH2) كهدف الربط (الشكل 1F). ثم أجرينا التحديد ابتمر الحمض النووي لايس استخدام دبة-NH2 كهدف الربط واتباع أسلوب المستندة إلى تجميد الهدف الكلاسيكي (الشكل 2). ف?...

Watch this Scientific Journal Video about Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor at JoVE.com

Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor

ويرد وضع بروتوكول للتحديد في المختبر وتوصيف الابتامرات الحمض النووي ملزم إستر حامض فثاليك الخاصة بالمجموعة. ويرد أيضا تطبيق ابتمر المحدد في أبتاسينسور الكهروكيميائية.

اختيار ابتمر الحمض النووي ملزم إستر حامض فثاليك وتوصيف وتطبيق أبتاسينسور الكهروكيميائية

Phthalic acid esters (PAEs) areone of the major groups of persistent organic pollutants. The group-specific detection of PAEs is highly desired due to the ...

phthalic-acid-ester-binding-dna-aptamer-selection-characterization

Research

JoVE Journal

Chemistry

9.8K Views.  Capital Normal University. ويرد وضع بروتوكول للتحديد في المختبر وتوصيف الابتامرات الحمض النووي ملزم إستر حامض فثاليك الخاصة بالمجموعة. ويرد أيضا تطبيق ابتمر المحدد في أبتاسينسور الكهروكيميائية.

Video: Phthalic Acid Ester-Binding DNA Aptamer Selection, Characterization, and Application to an Electrochemical Aptasensor

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

تصنيع أجهزة الاستشعار الكهروكيميائية الدنا للكشف Reagentless من الأحماض النووية والبروتينات والجزيئات الصغيرة

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Bioengineering

Mapping the Binding Site of an Aptamer on ATP Using MicroScale Thermophoresis

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an essential step in basic and applied science. MicroScale Thermophoresis (MST) is a sensitive biophysical method to obtain this important information. Relying on a physical effect called thermophoresis, which describes the movement of molecules through temperature gradients, this technology allows for the fast and precise determination of binding parameters in solution and allows the free choice of buffer conditions (from buffer to lysates/sera). MST uses the fact that an unbound molecule displays a different thermophoretic movement than a molecule that is in complex with a binding partner. The thermophoretic movement is altered in the moment of molecular interaction due to changes in size, charge, and hydration shell. By comparing the movement profiles of different molecular ratios of the two binding partners, quantitative information such as binding affinity (pM to mM) can be determined. Even challenging interactions between molecules of small sizes, such as aptamers and small compounds, can be studied by MST. Using the well-studied model interaction between the DH25.42 DNA aptamer and ATP, this manuscript provides a protocol to characterize aptamer-small molecule interactions. This study demonstrates that MST is highly sensitive and permits the mapping of the binding site of the 7.9 kDa DNA aptamer to the adenine of ATP.

MicroScale Thermophoresis (MST) is a sensitive technology to characterize aptamer-target interactions. This manuscript describes an MST protocol to characterize aptamer-small molecule interactions.

رسم خريطة الموقع ملزم لأبتمر على ATP عن طريق الميكروسكيل Thermophoresis

Characterization of molecular interactions in terms of basic binding parameters such as binding affinity, stoichiometry, and thermodynamics is an ...

Biochemistry

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

Bacterial Detection & Identification Using Electrochemical Sensors

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

كشف البكتيرية والاستدلال باستخدام مجسات الكهروكيميائية

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Electronic Tongue Generating Continuous Recognition Patterns for Protein Analysis

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of electronic tongues (eT) for protein analysis. By mixing a small number of simple and easily accessible molecules with different physicochemical properties, used as building blocks (BBs), in varying and controlled proportions and allowing the mixtures to self-assemble on the gold surface of a prism, an array of combinatorial surfaces featuring appropriate properties for protein sensing was created. In this way, a great number of cross-reactive receptors can be rapidly and efficiently obtained. By combining such an array of combinatorial cross-reactive receptors (CoCRRs) with an optical detection system such as surface plasmon resonance imaging (SPRi), the obtained eT can monitor the binding events in real-time and generate continuous recognition patterns including 2D continuous evolution profile (CEP) and 3D continuous evolution landscape (CEL) for samples in liquid. Such an eT system is efficient for discrimination of common purified proteins.

A novel approach is described for construction of electronic tongue (eT), which greatly simplifies the design and production of sensing materials, and allows the eT to generate continuous evolution profiles and landscapes for samples in liquid. The obtained eT is efficient for common protein analysis such as discrimination.

اللسان الإلكترونية توليد أنماط الاعتراف المستمر لتحليل البروتين

In current protocol, a combinatorial approach has been developed to simplify the design and production of sensing materials for the construction of ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

الفحص الحركي للنشاط النواسي باستخدام مجسات الحمض النووي

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Primer-Free Aptamer Selection Using A Random DNA Library

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified through an in vitro selection process called Systematic Evolution of Ligands by EXponential enrichment (SELEX) to recognize a wide variety of targets, from small molecules to proteins and other macromolecules 2-4. Aptamers have properties that are well suited for in vivo diagnostic and/or therapeutic applications: Besides good specificity and affinity, they are easily synthesized, survive more rigorous processing conditions, they are poorly immunogenic, and their relatively small size can result in facile penetration of tissues.
Aptamers that are identified through the standard SELEX process usually comprise ~80 nucleotides (nt), since they are typically selected from nucleic acid libraries with ~40 nt long randomized regions plus fixed primer sites of ~20 nt on each side. The fixed primer sequences thus can comprise nearly ~50% of the library sequences, and therefore may positively or negatively compromise identification of aptamers in the selection process 3, although bioinformatics approaches suggest that the fixed sequences do not contribute significantly to aptamer structure after selection 5. To address these potential problems, primer sequences have been blocked by complementary oligonucleotides or switched to different sequences midway during the rounds of SELEX 6, or they have been trimmed to 6-9 nt 7, 8. Wen and Gray 9 designed a primer-free genomic SELEX method, in which the primer sequences were completely removed from the library before selection and were then regenerated to allow amplification of the selected genomic fragments. However, to employ the technique, a unique genomic library has to be constructed, which possesses limited diversity, and regeneration after rounds of selection relies on a linear reamplification step. Alternatively, efforts to circumvent problems caused by fixed primer sequences using high efficiency partitioning are met with problems regarding PCR amplification 10.
We have developed a primer-free (PF) selection method that significantly simplifies SELEX procedures and effectively eliminates primer-interference problems 11, 12. The protocols work in a straightforward manner. The central random region of the library is purified without extraneous flanking sequences and is bound to a suitable target (for example to a purified protein or complex mixtures such as cell lines). Then the bound sequences are obtained, reunited with flanking sequences, and re-amplified to generate selected sub-libraries. As an example, here we selected aptamers to S100B, a protein marker for melanoma. Binding assays showed Kd s in the 10-7 - 10-8 M range after a few rounds of selection, and we demonstrate that the aptamers function effectively in a sandwich binding format.

SELEX protocols comprise multiple rounds of selection, each of which require regeneration of bound ligands, which in turn require fixed primer sequences flanking the random library regions. These fixed primer sequences can interfere with the selection process (false positives and negatives). Here we present a primer-free protocol.

خالية من التحديد التمهيدي Aptamer باستخدام مكتبة DNA عشوائية

Aptamers are highly structured oligonucleotides (DNA or RNA) that can bind to targets with affinities comparable to antibodies 1. They are identified ...

Biology

Determining the Thermodynamic and Kinetic Association of a DNA Aptamer and Tetracycline Using Isothermal Titration Calorimetry

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for application. Due to the drastic differences between the aptamer and small molecules, scientists need to put much effort into characterizing their binding properties. Isothermal Titration Calorimetry (ITC) is a powerful approach for this purpose. ITC goes beyond determining disassociation constants (Kd) and can provide the enthalpy changes and binding stoichiometry of the interaction between two molecules in the solution phase. This approach conducts continuous titration using label-free molecules and records released heat over time upon the binding events produced by each titration, so the process can sensitively measure the binding between macromolecules and their small targets. Herein, the article introduces a step-by-step procedure of the ITC measurement of a selected aptamer with a small target, tetracycline. This example proves the versatility of the technique and its potential for other applications.

The present protocol describes the use of Isothermal Titration Calorimetry (ITC) to analyze the association and dissociation kinetics of the binding between a DNA aptamer and tetracycline, including sample preparation, running standards and samples, and interpreting the resulting data.

تحديد الارتباط الديناميكي الحراري والحركي لأبتامر الحمض النووي والتتراسيكلين باستخدام كالوريمتر المعايرة بالتحليل الحجمي متساوي الحرارة

The determination of binding affinity and behavior between an aptamer and its target is the most crucial step in selecting and using an aptamer for ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

تطوير Biosensor الكهروكيميائية الحمض النووي للكشف عن مسببات الأمراض المنقولة عن طريق الأغذية

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

Ultrasensitive Detection of Biomarkers by Using a Molecular Imprinting Based Capacitive Biosensor

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the detection of biomarkers, contaminants, and other molecules of interest. A commonly used technique for this purpose is the Enzyme-linked Immunosorbent Assay (ELISA), where often one antibody is directed towards a specific target molecule, and a second labeled antibody is used for the detection of the primary antibody, allowing for the absolute quantification of the biomolecule under study. However, the usage of antibodies as recognition elements limits the robustness of the method; as does the need of using labeled molecules. To overcome these limitations, molecular imprinting has been implemented, creating artificial recognition sites complementary to the template molecule, and obsoleting the necessity of using antibodies for initial binding. Further, for even higher sensitivity, the secondary labeled antibody can be replaced by biosensors relying on the capacitance for the quantification of the target molecule. In this protocol, we describe a method to rapidly and label-free detect and quantitate low-abundant biomolecules (proteins and viruses) in complex samples, with a sensitivity that is significantly better than commonly used detection systems such as the ELISA. This is all mediated by molecular imprinting in combination with a capacitance biosensor.

Here, we present a protocol for the detection and quantification of low abundant molecules in complex solutions using molecular imprinting in combination with a capacitance biosensor.

الكشف عن أولتراسينسيتيفي للمؤشرات الحيوية باستخدام طابعة جزيئية أساس Biosensor سعوية

The ability to detect and quantitate biomolecules in complex solutions has always been highly sought-after within natural science; being used for the ...

Immunology and Infection

Aptamer-Based Target Detection Facilitated by a 3-Stage G-Quadruplex Isothermal Exponential Amplification Reaction

Aptamers are target-recognition molecules that bind with high affinity and specificity. These characteristics can be leveraged to control other molecules with signal-generation capability. For the system described herein, target recognition through an aptameric domain, Stem II of a modified hammerhead ribozyme, activates the self-cleaving ribozyme by stabilizing the initially unstructured construct. The cis-cleaving RNA acts at the junction of Stem III and Stem I, creating two cleavage products. The longer cleavage product primes an isothermal exponential amplification reaction (EXPAR) of the two similar catalytically active G-quadruplexes. Those resulting amplification products catalyze peroxidase reduction, which is coupled to the reduction of a colorimetric substrate with an output that the naked eye can detect. The 3-part system described in the present study improves detection modalities such as enzyme-linked immunosorbent assays (ELISAs) by producing a visually detectable signal for indicating the presence of as low as 0.5 &#181;M theophylline in as little as 15 min.

The present protocol demonstrates the use of a fast, 3-stage, aptamer-based exponential amplification assay to detect targets. Sample preparation, signal amplification, and color development are covered to implement this system to recognize the presence of theophylline over that of caffeine.

يتم تسهيل الكشف عن الهدف القائم على Aptamer بواسطة تفاعل تضخيم أسي متساوي الحرارة G-quadruplex من 3 مراحل

Aptamers are target-recognition molecules that bind with high affinity and specificity. These characteristics can be leveraged to control other molecules ...

اختيار ابتمر الحمض النووي ملزم إستر حامض فثاليك وتوصيف وتطبيق أبتاسينسور الكهروكيميائية

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