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

우리는 국립 자연 과학 재단 (21675112), 베이징 교육 위원회 (KZ201710028027)와 Yanjing 젊은 학자 프로그램의 수도 사범 대학의 과학 및 기술 계획의 프로젝트에서에서 재정 지원에 대 한 감사.

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저자는 경쟁 금융 관심을 선언합니다.

Aptamers의 뛰어난 이점 중 하나입니다 vivo에서 immunoreactions를 통해 항 체를 생성 하는 동안 그들은 체 외에서 방법을 SELEX 통해 식별 됩니다. 따라서, 항 체는 생리 적 조건에 제한 하는 반면 aptamers 잘 설계 된 실험 조건에서 원하는 대상으로 선택할 수 있습니다.
무료 시퀀스에서 바운드 시퀀스의 분리를 촉진 하기 위하여 몇 가지 수정 된 SELEX 최근 보고 되었습니다,...

급속 한 경제 발전, 함께 산업화, 그리고 도시 건설, 환경 오염의 가속 그 어느 때 보다 더 심각 하다. 일반적인 환경 오염 물질 중 금속 이온, 독 소, 항생제, 농약, 내 분 비 분리기와 지속적인 유기 오염 물질 (Pop)을 포함합니다. 금속 이온과 독 소, 외 다른 오염 물질은 작은 분자는 꽤 congeners의 다양 한 구성 됩니다. 가장 독성 Pop 비페닐 (PCBs), 다 환 방향족 탄화수소 (PAHs), 폴 리 브롬 화 비페닐 에테르 (PBDEs), polychlorinated dibenzo-p-다이옥신 (PCDDs), polychlorinated dibenzofuran (PCDFs)를 포함 하는 예를 들어 고 phthalic 산 에스테 르 (PAEs)1,-2모든 많은 congeners의 구성. 작은 분자 검출 크로마토그래피/질량 분석 기반 기술을 응용 프로그램3,4,,56의 그들의 다양성 때문에 의해 주로 수행 되었습니다. 사이트 검색에 대 한 항 체 기반 방법을 최근 개발된7,,89되었습니다 합니다. 그러나, 이러한 방법은 특정 느리기에 대 한 매우 구체적인 이기 때문에, 여러 테스트를 수행 합니다. 더 심각한 것은 소설 congeners 성장 빠른 시간에 그들의 항 체를 생성할 수 없습니다. 따라서, 한 테스트에 모든 congeners의 총 레벨 모니터링 하 그룹 관련 바이오 센서의 개발 환경 오염 상태를 평가 하기 위한 귀중 한 통계를 제공할 수 있습니다.
최근, 핵 산 aptamers 널리 적용 된 다양 한 바이오 센 싱 플랫폼 다양 한 이온과 단백질 및 세포10,11 작은 분자에서 대상 인식의 그들의 기능 때문에 인식 요소 ,12. Aptamers는 시험관에 의해 호출 된 메서드 ligands의 체계적인 발전 지 수 농축 (SELEX)13,14을 통해 식별 됩니다. SELEX는 약 1014-1015 시퀀스 포함 임의의 합성 단일 가닥 oligonucleotide 도서관으로 시작 합니다. 무작위 라이브러리의 크기는 RNA 또는 DNA 후보 구조의 다양성을 보장합니다. 전형적인 SELEX 프로세스 라이브러리는 높은 친 화력 및 특이성 대상 시퀀스에 농축 될 때까지 농축의 여러 라운드로 구성. 최종 농축된 수영장 다음 시퀀스와 분리 상수 (Kd) 및 간섭 물질과 같은 다른 기술에 의해 결정 하는 잠재력에 대 한 선택 필터 바인딩, 친화성 크로마토그래피, 표면 플라스몬 공명 (SPR), 등. 15
매우 빈약한 물 가용성과 표면 동원 정지에 대 한 기능 그룹의 부족, pop aptamer 선택은 이론적으로 어렵습니다. SELEX에 대 한 중요 한 발전 aptamers 발견 가속화 했습니다. 그러나, 팝에 대 한 그룹별 aptamers의 선택은 하지 아직 알려졌다. 지금까지 특정 느리기에 대 한 높은 특이성으로 PCB 바인딩 DNA aptamers만 확인 된16되었습니다. PAEs는 주로 폴 리 염화 비닐 물자, 탄력 있는 플라스틱, 따라서 가소제 역할 하드 플라스틱에서 폴 리 염화 비닐을 변경에 사용 됩니다. 일부 PAEs 내 분 비 분리기로 확인 되었습니다, 심각한 손상을 일으킬 수 간 및 신장 기능, 남성 정자의 운동 성 감소 하 고 비정상적인 정자 형태와 고환 암17발생할 수 있습니다. 화합물-도 그룹 전용 PAE 바인딩 aptamers 보고 되었습니다.
이 작품의 목표 그룹-특정 DNA aptamers PAEs, 팝의 대표 그룹 등 매우 소수 작은 분자를 선택 하기 위한 대표적인 프로토콜을 제공 하는 것입니다. 우리는 또한 환경 오염 감지를 위한 선택 된 aptamer의 응용 프로그램을 보여 줍니다. 이 프로토콜 다른 소수 작은 분자의 aptamer 발견에 대 한 지침과 통찰력을 제공합니다.

<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

지속적인 유기 오염 물질의 주요 그룹의 phthalic 산 에스테 르 (PAEs) areone. PAEs의 그룹 전용 탐지는 congeners의 급속 한 성장으로 인해 매우 원한다. 점점 더 인식 요소 바이오 센서 플랫폼에 적용 된 DNA aptamers 하지만 선택 aptamers PAEs, 등 매우 소수 성 분자 표적으로 보고 거의. 이 작업 그룹-특정 DNA aptamers PAEs 선택 하도록 설계 된 비드 기반 방법을 설명 합니다. 아미노 그룹 공업화 틸 프 탈 레이트 (DBP-NH2) 앵커 대상 합성 했 고 에폭시 활성화 agarose 구슬에 움직일 immobilization 매트릭스의 표면에서 phthalic 에스테 르 그룹의 표시를 허용 하 고 따라서 그룹 전용 바인더의 선택. 자력 분리와 결합 하는 정량적 중 합 연쇄 반응에 따라 aptamer 후보자의 분리 상수 하 고. 상대 선호도 다른 PAEs에 aptamers의 선택도 경쟁 분석 실험에 의해 결정 되었다 aptamer 후보 DBP NH2 미리 경계 했다 연결 된 자석 구슬, 가진 외피에 상쾌한에 발표 테스트 PAEs 또는 다른 잠재적인 간섭 물질입니다. 경쟁 분석 결과 표면 동원 정지에 대 한 기능 그룹이 했다 PAEs 중 손쉬운 선호도 비교를 제공 하기 때문에 적용 되었다. 마지막으로 우리 전기 aptasensor의 제작을 시연 하 고 bis(2-ethylhexyl) 프 탈 레이트의 磁 및 선택적 검출을 위해 사용. 이 프로토콜 다른 소수 작은 분자의 aptamer 발견에 대 한 통찰력을 제공합니다.

우리는 설계 및 아미노 그룹 공업화 틸 프 탈 레이트 (DBP-NH2) 앵커 대상 (그림 1 층)으로 합성. 우리는 다음 PAEs 클래식 대상 immobilization 기반 방법 (그림 2)에 따라는 DBP NH2 앵커 대상으로 사용 하 여 DNA aptamer 선택을 수행 합니다. 각 라운드에서 파일럿 PCR PCR (그림 3)의 주기 수를 최적화 하는 ...

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

생체 외에서 선택 및 그룹 관련 phthalic 산 에스테 르-바인딩 DNA aptamers의 특성화에 대 한 프로토콜 제공 됩니다. 전기 aptasensor에서 선택 된 aptamer의 응용 프로그램이 포함 되어 있습니다.

Phthalic 산 에스테 르-바인딩 DNA Aptamer 선택, 특성, 및 전기 화학 Aptasensor 응용 프로그램

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 조회수.  Capital Normal University. 생체 외에서 선택 및 그룹 관련 phthalic 산 에스테 르-바인딩 DNA aptamers의 특성화에 대 한 프로토콜 제공 됩니다. 전기 aptasensor에서 선택 된 aptamer의 응용 프로그램이 포함 되어 있습니다.

비디오: 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.

핵산, 단백질과 작은 분자의 Reagentless 감지를위한 전기 -의 DNA Biosensors의 제작

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. ...

연구

생체공학

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.

마이크로 열 영동을 사용하여 ATP에 앱 타머의 결합 부위를 매핑

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

생화학

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.

전기 화학 센서를 사용하여 세균의 검출 및 식별

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

A Microfluidic-based Electrochemical Biochip for Label-free DNA Hybridization Analysis

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration of pre-processing steps. Utilizing these devices towards the analysis of DNA hybridization events is important because it offers a technology for real time assessment of biomarkers at the point-of-care for various diseases. However, when the device footprint decreases the dominance of various physical phenomena increases. These phenomena influence the fabrication precision and operation reliability of the device. Therefore, there is a great need to accurately fabricate and operate these devices in a reproducible manner in order to improve the overall performance. Here, we describe the protocols and the methods used for the fabrication and the operation of a microfluidic-based electrochemical biochip for accurate analysis of DNA hybridization events. The biochip is composed of two parts: a microfluidic chip with three parallel micro-channels made of polydimethylsiloxane (PDMS), and a 3 x 3 arrayed electrochemical micro-chip. The DNA hybridization events are detected using electrochemical impedance spectroscopy (EIS) analysis. The EIS analysis enables monitoring variations of the properties of the electrochemical system that are dominant at these length scales. With the ability to monitor changes of both charge transfer and diffusional resistance with the biosensor, we demonstrate the selectivity to complementary ssDNA targets, a calculated detection limit of 3.8 nM, and a 13% cross-reactivity with other non-complementary ssDNA following 20 min&#160;of incubation. This methodology can improve the performance of miniaturized devices by elucidating on the behavior of diffusion at the micro-scale regime and by enabling the study of DNA hybridization events.

We present a microfluidic-based electrochemical biochip for DNA hybridization detection. Following ssDNA probe functionalization, the specificity, sensitivity, and detection limit are studied with complementary and non-complementary ssDNA targets. Results illustrate the influence of the DNA hybridization events on the electrochemical system, with a detection limit of 3.8 nM.

라벨없는 DNA의 혼성화 분석 용 미세 유체 기반 전기 바이오칩

Miniaturization of analytical benchtop procedures into the micro-scale provides significant advantages in regards to reaction time, cost, and integration ...

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.

단백질 분석을위한 지속적인 인식 패턴을 생성하는 전자 혀

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

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.

랜덤 DNA 라이브러리를 사용하여 뇌관 - 무료 Aptamer 선택

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

생물학

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.

등온 적정 열량계를 사용하여 DNA 압타머와 테트라사이클린의 열역학적 및 동역학적 연관성 결정

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.

Foodborne 병원 체를 검출 하는 전기 화학 DNA 바이오 센서의 개발

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.

분자 각 인을 사용 하 여 생체의 磁 검출 용량 성 바이오 센서 기반

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

면역학 및 감염병학

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.

3단계 G-Quadruplex 등온 지수 증폭 반응에 의해 촉진되는 Aptamer 기반 표적 검출

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