이 작품은 음식과 농업에 대한 USDA 국립 연구소, 국립 경쟁 보조금 프로그램, 국가 연구 이니셔티브 프로그램 71.1 FY 2007에 의해 부분적으로 자금 지원 알레르기 및 감염성 질환 그랜트 번호 2007-35503-18392, 보건 국립 연구소, 연구소 등 , R33AI94514-03.

1 회전 마약 준비 <ol><li> 이러한 계산에서 생체 고분자 분말의 수분 함량을 고려 반드시 생체 고분자의 농도 범위 (/ V 와트, 30 %, 0.1 %)을 조사하도록 준비한다. 각각의 농도를 들어, 50 ㎖의 시험관에 생체 고분자 (전분 또는 풀루 란) 분말을 무게. 수성 디메틸 설폭 사이드 (DMSO) 용액과 교반 막대를 추가합니다. </li><li> 자석 교반기의 핫 플레이트에 일정한 교반 끓는 물에 튜브를 놓습니다. </li><li> 약 1 시간 후, 열을 끄고 분산액을 실온으로 냉각되도록. 분산 물을 유동 학적 검사와 전기 방사를위한 준비가되어있다. </li></ol> 2 꾸준한 전단 유변학 <ol><li> 레오 미터를 따뜻하게하고 20 ° C에서 무대 온도를 설정합니다. 프로브 (사용 25mm 콘) 및 스테이지 (판) 사이의 격차를 보정합니다. </li><li> 상기 스테이지의 중심 상 생체 고분자 분산액 0.41 ㎖의 부하 positi를 설정하는 프로브를 낮출(25mm 콘에 대한 0.053 mm 간격)에. 분산이 균등하게 간격 내에서 확산 있는지 확인합니다. </li><li> 다음 실험 매개 변수 유변학 테스트를 수행 스윕 모드 : 로그인, 초기 속도 : 100 초 -1, 최종 속도 : 0.1 초 -1, 십 년간 당 포인트 : 10, 측정 전에 지연 : 5 초, 시간을 측정 : 10 초, 측정 기준과 방향 : 두 (시계 및 반 시계 방향 모두). </li><li> 시험을 전기 방사에 적합한 분산 농도를 추정하기 위해 유동 학적 데이터를 분석 할 수 있습니다. <ol><li> 중합체 농도의 함수로서 전단 율 대 겉보기 전단 점도 플롯. 0.1 초에서 겉보기 점도 (낮은 전단 속도 데이터가 신뢰할 낮은 농도에서 예) 실제 또는 추정 값을 기준으로 각 플로우 곡선, 대략 제로 전단 점도, η 0, 용 - 1. </li><li> 특정 점도를 계산 η 특검팀 = (η 0 - η들)η S는 용매의 점도 / s의 η. </li><li> 농도의 함수로서 고유 점도 플롯. semidilute unentangled 얽힌 정권을 확인합니다. semidilute unentangled 정권 작은 기울기 저농도 끝에서 시작하고, semidilute 교락 정권 unentangled 정권 다음 더 큰 경사를 갖는다. 두 정권에 맞추기 지수 법칙 회귀 모델. 전력 값은 로그 - 로그 플롯에 semidilute unentangled 낙합 정권과 경사면 (농도 의존성)이다. 이 장착 선의 교차점 얽힘 농도, C 전자이다. </li></ol></li></ol> 3 전기 방사 매개 변수 변화 <ol><li> 예를 들면, 15 %도 1에 도시 된 바와 같이. 전기 방사 셋업 조립 적절한 조성물의 분산액으로 주사기를 부하 (/ w를 v) 100 % DMSO에서, 주사기 펌프 상 전분 또는 풀루. 멋 부리 (고전압 와이어 클립바늘에 필자). 화장실에 접지 와이어 (음)을 담가 땅에 순수 에탄올을 포함하는 응고 욕을 연결합니다. 주사기 바늘과 응고 욕 사이의 거리를 조정하기 위해 랩 잭을 사용. 전기 방사 한 후 섬유 매트를 수집 조에서 금속 메쉬 담가. </li><li> 다음 매개 변수 범위의 생체 고분자를 스핀 : 이송 속도 0.1 ~ 0.4 ㎖ / 시간, 5-10cm 회전 거리와 전압에서 0 ~ 15 kV의에. <ol><li> 5cm의 회전 거리를 시작합니다. 제 이송 속도 (0.1 ㎖ / h)를 위해, 바늘 끝에서 압출 분산액의 형상으로 서서히 영 V. 결제주의로부터의 전압을 램프 업 및 적하 분산액 가속하고 기다란 때주의한다. </li><li> 작은 제트 솔루션의 electrospinnability를 나타내는 드롭 표면에서 시작되는 전압을합니다. 연속 제트가있는 경우, 시작되는 전압을 기록한다. </li><li> 세 paramete 각각에 대한 전체 범위를 검토RS 및 참고 성공적인 방사 조건. 끝에서 연속 제트있을 때에 만 섬유를 수집. </li></ol></li><li> 수집 몇 분 후, 순수 에탄올로 섬유 매트를 헹군다. 진공 상태에서 건조를 포함하는 데시 케이 터에 섬유 매트를 놓습니다. </li><li> 완전한 특성에 대한 각 생체 고분자의 농도에 대해 반복합니다. </li></ol><img alt="그림 1" fo:content-width="5in" src="/files/ftp_upload/51933/51933fig1highres.jpg" width="500" /> 전기 - 습식 방사 셋업도 1은 개략도. 생체 고분자 분산액을 주사기 펌프로부터 압출된다. 고전압 DC 전원이 무딘 바늘 근거 응고 욕에 높은 전압을 제공한다. 바늘 끝에서 폴리머 제트는 직선 경로를 통해 이동 한 다음 (일명 불안정성을 흔든다) 빠른 휘핑 경로를 개발하고 있습니다. (4) 형태 학적 특성<ol><li> 건조 섬유 매트의 조각을 잘라 탄소 테이프를 사용 SEM 스텁에 그것을 고정시킨다. </li><li> SEM 악기로 샘플 스텁을로드하고 분석을 위해 이미지를 얻을 수 있습니다. </li></ol>

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저자들은 공개 아무것도 없다는 것을 선언합니다.

레올 종래 섬유 방적을 포함하고 13 전기 방사 중합체의 처리를 연구하는 필수적인 도구이다. 정상 전단 유동 학적 연구, 고분자 형태 및 다른 용매에서의 상호 작용에서 해결 될 수 있습니다 (그림 2와 그림 3). 생체 고분자 분자들이 서로 중첩하기에 충분한 높은 농도에서, 그들의 농도 의존성은 좋은 용매 3,4 다른 중합체의보고 된 값과 잘 일치이었다 약 1.4 (도 ...

전기 방사는 다양한 물질로부터 나노 크기의 연속 마이크로 섬유를 제조 할 수있는 기술이다. 그것은 학문과 산업의 관심 일 증가 얻고있다. 설치 및 전기 방사의 연습은 간단 보이지만, 섬유 속성을 electrospinnability을 예측하고 제어 할 수있는 능력은 과제로 남아. 그 이유는 전기 방사 (2) 및 프로세스, 특히 섬유에 의해 이동 경로 일 것이다 혼란을 좌우하는 여러 가지 요인이 있다는 사실에 놓여있다. 종종 경험 &quot;보고 요리 -와 -&quot;접근 방식은 잠재적 electrospinnable 물질을 선별하는 데 사용됩니다. 그러나, 전기 방사를 더 잘 제어 및 합성 섬유의 특성, 필요 electrospinnability을 지배하는 메커니즘에 대한 이해를 돕기 있습니다. 몇몇 연구자들은 원액에 중합체의 분자의 얽힘이 된 essentia 것을 발견성공적인 전기 방사 3 - 5 리터의 필수 조건. 레올 로지 고분자 분산 분자 형태와 상호 작용을 조사 할 수있는 강력한 도구입니다. 예를 들어는 맥키 등. 클로로포름 / 디메틸 테레 프탈레이트 (7/3, v / v)로 함유하는 용매에 선형의 분자 형태 및 분 지형 폴리 (에틸렌 테레 프탈레이트 - 코 - 에틸렌 이소 프탈레이트) 공중 합체를 조사하고, 중합체 농도가 2-2.5x이어야 한 것으로 판단 성공적인 전기 방사 4 얽힘 농도. 때문에 생분해 성, 생체 적합성, 그리고 힘? 힘을 자신의 합성 대응 개성를에서 자신의 장점의 바이오 폴리머에서 섬유에서 현재 새롭게 관심이 있습니다. 그러나 실무자 열 처리 및 열등한 기계적 특성에 구조적 복잡성, 어려움에서 일반적으로 발생하는 많은 도전에 직면. 식물 조직에서 발견되는 전분은, 아몬이며g 지구상에서 가장 풍부하고 저렴한 바이오 폴리머. 순수 전분 섬유는 습식 전기 방사 장치는 최근 6 바와 이용하여 제조 하였다. 풀루 란 특정 박테리아에 의해 세포 외로 생산 선형 다당류이다. (1 → 4)와 (1 → 6)의 정규 교대는 glucosidic 채권은 능력 7,8를 형성하는 우수한 섬유 / 필름 포함 풀루 란의 여러 가지 독특한 속성에 대한 책임을 것으로 생각됩니다. 수성 분산액에서 풀루 섬유는 전기 방사 9,10 연구자들에 의해보고되었다. 이전 출판물에서이 바이오 폴리머, 전분 11, 풀루 란 (12)의 electrospinnability이 논의되고있다. 이 보고서는이 두 가지 생체 고분자의 electrospinnability의 조사에 유동 학적 원리를 활용하기위한 프로토콜을 시연에 초점을 맞추고 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="762">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:253px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:175px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Gelose 80 starch</td>
			<td style="width:253px;">Ingredion</td>
			<td style="width:119px;"></td>
			<td>Used as it is</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pullulan</td>
			<td style="width:253px;">Hayashibara Co. Ltd</td>
			<td style="width:119px;"></td>
			<td>Used as it is</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Dimethyl Sulfoxide</td>
			<td style="width:253px;">BDH Chemicals</td>
			<td style="width:119px;">BDH1115-4LP</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:216px;">Ethanol</td>
			<td style="width:253px;">VWR International</td>
			<td style="width:119px;">89125-172</td>
			<td>200 proof</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:253px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rheometer</td>
			<td style="width:253px;">TA Instruments</td>
			<td style="width:119px;">ARES&#160;</td>
			<td>50 mm cone and plate geometry</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe (10 mL)</td>
			<td style="width:253px;">Becton, Dickinson and Company</td>
			<td style="width:119px;">309604</td>
			<td>Syringe with Luer-Lok&#174; Tip</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:216px;">High voltage generator</td>
			<td style="width:253px;">Gamma High Voltage Research, Inc.</td>
			<td style="width:119px;">ES40P</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe pump</td>
			<td style="width:253px;">Hamilton Company</td>
			<td style="width:119px;">81620</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Environmental scanning electron microscope</td>
			<td style="width:253px;">FEI Company</td>
			<td style="width:119px;">Quanta 200</td>
			<td>for starch fibers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Environmental scanning electron microscope</td>
			<td style="width:253px;">Phenom-World</td>
			<td style="width:119px;">Phenom G2 Pro</td>
			<td>for pullulan fibers</td>
		</tr>
	</tbody>
</table>

molecular entanglement and electrospinnability of biopolymers

전기 방사는 다양한 물질로부터 나노 섬유를 제조하는 마이크로 매혹적인 기술이다. 생체 고분자를 들어, 방사 도프의 성분 중합체의 분자의 얽힘이 성공적인 전기 방사를위한 필수적인 전제 조건 인 것으로 밝혀졌다. 유변학은 생체 고분자의 분자 형태와 상호 작용을 조사 할 수있는 강력한 도구입니다. 이 보고서에서, 우리는 디메틸 설폭 사이드 (DMSO) / 물 분산액으로부터,이 생체 고분자, 전분 및 풀루 란 electrospinnability을 평가 레올을 이용하는 프로토콜을 설명한다. 미크론 범위 미크론의 평균 직경을 갖는 잘 형성된 전분 및 풀루 섬유가 얻어졌다. Electrospinnability 형성 섬유의 시각 및 현미경 관찰에 의해 평가 하였다. 자신의 electrospinnability에 분산의 유변학 적 특성에 대한 상관 관계를 우리는 모든 당선자에 영향을 분자 형태, 분자 얽힘, 및 전단 점도 입증rospinning. 유 동학은 용매 시스템 선택 및 공정 최적화에 유용 할뿐만 아니라 분자 수준에서 섬유 형성의 메커니즘을 이해하지 않다.

생체 고분자 농도 및 용매 DMSO 농도의 함수로서 생체 고분자 분산액의 유동 곡선을 구 하였다. 두 대표적인 수치는 순수한 DMSO 용매에서의 농도의 함수로서 전분 (도 2A) 및 플루 란 (도 2b)의 유동 곡선을 나타낸다. 특정 점도는 생체 고분자 농도 (전분 그림 3a 및 풀루 란 그림 3B) 역모를 꾸몄다되었다. 이들 플롯에서, 엉킴 농도 semidilute unentangled 및 ...

Watch this Scientific Journal Video about Molecular Entanglement and Electrospinnability of Biopolymers at JoVE.com

Molecular Entanglement and Electrospinnability of Biopolymers

전기 방사는 다양한 물질에서 나노 스케일로 미세 섬유를 제조하는데 사용 매력적인 방법이다. 방사 도프의 성분 중합체의 분자의 얽힘이 성공적인 전기 방사에 필수적이다. 우리는이 생체 고분자, 전분 및 풀루 란 electrospinnability을 평가 레올 이용하는 프로토콜을 제시한다.

분자 얽힘과 생체 고분자의 Electrospinnability

Electrospinning is a fascinating technique to fabricate micro- to nano-scale fibers from a wide variety of materials. For biopolymers, molecular ...

molecular-entanglement-and-electrospinnability-of-biopolymers

Research

JoVE Journal

Bioengineering

13.6K 조회수.  Pennsylvania State University.  전기 방사는 다양한 물질에서 나노 스케일로 미세 섬유를 제조하는데 사용 매력적인 방법이다. 방사 도프의 성분 중합체의 분자의 얽힘이 성공적인 전기 방사에 필수적이다. 우리는이 생체 고분자, 전분 및 풀루 란 electrospinnability을 평가 레올 이용하는 프로토콜을 제시한다. 

비디오: Molecular Entanglement and Electrospinnability of Biopolymers

Fluorescence Lifetime Imaging of Molecular Rotors in Living Cells

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular events. Viscosity is one of the key parameters affecting the diffusion of molecules and proteins, and changes in viscosity have been linked to disease and malfunction at the cellular level.1-3 While methods to measure the bulk viscosity are well developed, imaging microviscosity remains a challenge. Viscosity maps of microscopic objects, such as single cells, have until recently been hard to obtain. Mapping viscosity with fluorescence techniques is advantageous because, similar to other optical techniques, it is minimally invasive, non-destructive and can be applied to living cells and tissues.
Fluorescent molecular rotors exhibit fluorescence lifetimes and quantum yields which are a function of the viscosity of their microenvironment.4,5 Intramolecular twisting or rotation leads to non-radiative decay from the excited state back to the ground state. A viscous environment slows this rotation or twisting, restricting access to this non-radiative decay pathway. This leads to an increase in the fluorescence quantum yield and the fluorescence lifetime. Fluorescence Lifetime Imaging (FLIM) of modified hydrophobic BODIPY dyes that act as fluorescent molecular rotors show that the fluorescence lifetime of these probes is a function of the microviscosity of their environment.6-8 A logarithmic plot of the fluorescence lifetime versus the solvent viscosity yields a straight line that obeys the F&ouml;rster Hoffman equation.9 This plot also serves as a calibration graph to convert fluorescence lifetime into viscosity.
Following incubation of living cells with the modified BODIPY fluorescent molecular rotor, a punctate dye distribution is observed in the fluorescence images. The viscosity value obtained in the puncta in live cells is around 100 times higher than that of water and of cellular cytoplasm.6,7 Time-resolved fluorescence anisotropy measurements yield rotational correlation times in agreement with these large microviscosity values. Mapping the fluorescence lifetime is independent of the fluorescence intensity, and thus allows the separation of probe concentration and viscosity effects. 
In summary, we have developed a practical and versatile approach to map the microviscosity in cells based on FLIM of fluorescent molecular rotors.

Fluorescence Lifetime Imaging (FLIM) has emerged as a key technique to image the environment and interaction of specific proteins and dyes in living cells. FLIM of fluorescent molecular rotors allows mapping of viscosity in living cells.

생활 세포의 분자 Rotors의 형광 수명 이미징

Diffusion is often an important rate-determining step in chemical reactions or biological processes and plays a role in a wide range of intracellular ...

연구

생체공학

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular machines centers on devices capable of directional motion, i.e. molecular motors, and on their scaled-down mechanical parts (wheels, axels, pendants etc) 7-9. This imitates the macro-machines, even though the physical properties essential for these devices, such as inertia and momentum conservation, are not usable in the nanoworld environments 10. Alternative designs, which do not follow the mechanical macromachines schemes and use mechanisms developed in the evolution of biological molecules, can take advantage of the specific conditions of the nanoworld. Besides, adapting actual biological molecules for the purposes of nano-design reduces potential dangers the nanotechnology products may pose. Here we demonstrate the assembly and application of one such bio-enabled construct, a semi-artificial molecular device which combines a naturally-occurring molecular machine with artificial components. From the enzymology point of view, our construct is a designer fluorescent enzyme-substrate complex put together to perform a specific useful function. This assembly is by definition a molecular machine, as it contains one 12. Yet, its integration with the engineered part - fluorescent dual hairpin - re-directs it to a new task of labeling DNA damage12.
Our construct assembles out of a 32-mer DNA and an enzyme vaccinia topoisomerase I (VACC TOPO). The machine then uses its own material to fabricate two fluorescently labeled detector units (Figure 1). One of the units (green fluorescence) carries VACC TOPO covalently attached to its 3'end and another unit (red fluorescence) is a free hairpin with a terminal 3'OH. The units are short-lived and quickly reassemble back into the original construct, which subsequently recleaves. In the absence of DNA breaks these two units continuously separate and religate in a cyclic manner. In tissue sections with DNA damage, the topoisomerase-carrying detector unit selectively attaches to blunt-ended DNA breaks with 5'OH (DNase II-type breaks)11,12, fluorescently labeling them. The second, enzyme-free hairpin formed after oligonucleotide cleavage, will ligate to a 5'PO4 blunt-ended break (DNase I-type breaks)11,12, if T4 DNA ligase is present in the solution 13,14 . When T4 DNA ligase is added to a tissue section or a solution containing DNA with 5'PO4 blunt-ended breaks, the ligase reacts with 5'PO4 DNA ends, forming semi-stable enzyme-DNA complexes. The blunt ended hairpins will interact with these complexes releasing ligase and covalently linking hairpins to DNA, thus labeling 5'PO4 blunt-ended DNA breaks.
This development exemplifies a new practical approach to the design of molecular machines and provides a useful sensor for detection of apoptosis and DNA damage in fixed cells and tissues.

In vitro Assembly of Semi-artificial Molecular Machine and its Use for Detection of DNA Damage

We demonstrate the assembly and application of a molecular-scale device powered by a topoisomerase protein. The construct is a bio-molecular sensor which labels two major types of DNA breaks in tissue sections by attaching two different fluorophores to their ends.

DNA 손상 감지를위한 세미 인공 분자 기계 및 이용 체외 조립에

Naturally occurring bio-molecular machines work in every living cell and display a variety of designs 1-6. Yet the development of artificial molecular ...

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

Biomarker 디스커버리를위한 Mesoporous 실리카 박막의 낮은 분자량의 단백질 농축

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

Flexural Rigidity Measurements of Biopolymers Using Gliding Assays

Microtubules are cytoskeletal polymers which play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires microtubules that are stiff and straight enough to span a significant fraction of the cell diameter. As a result, the microtubule persistence length, a measure of stiffness, has been actively studied for the past two decades1. Nonetheless, open questions remain: short microtubules are 10-50 times less stiff than long microtubules2-4, and even long microtubules have measured persistence lengths which vary by an order of magnitude5-9.
	Here, we present a method to measure microtubule persistence length. The method is based on a kinesin-driven microtubule gliding assay10. By combining sparse fluorescent labeling of individual microtubules with single particle tracking of individual fluorophores attached to the microtubule, the gliding trajectories of single microtubules are tracked with nanometer-level precision. The persistence length of the trajectories is the same as the persistence length of the microtubule under the conditions used11. An automated tracking routine is used to create microtubule trajectories from fluorophores attached to individual microtubules, and the persistence length of this trajectory is calculated using routines written in IDL.
	This technique is rapidly implementable, and capable of measuring the persistence length of 100 microtubules in one day of experimentation. The method can be extended to measure persistence length under a variety of conditions, including persistence length as a function of length along microtubules. Moreover, the analysis routines used can be extended to myosin-based acting gliding assays, to measure the persistence length of actin filaments as well.

A method to measure the persistence length or flexural rigidity of biopolymers is described. The method uses a kinesin-driven microtubule gliding assay to experimentally determine the persistence length of individual microtubules and is adaptable to actin-based gliding assays.

글라이딩 Assays를 사용 Biopolymers의 굽힘 강성 측정

Microtubules are cytoskeletal polymers which  play a role in cell division, cell mechanics, and intracellular transport. Each of these functions requires ...

Characteristics of Precipitation-formed Polyethylene Glycol Microgels Are Controlled by Molecular Weight of Reactants

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer several advantages over traditional microsphere fabrication techniques. Contrary to emulsion, suspension, and dispersion techniques, microgels formed by precipitation are of uniform shape and size, i.e.&#160;low polydispersity index, without the use of organic solvents or stabilizers. The mild conditions of the precipitation reaction, customizable properties of the microgels, and low viscosity for injections make them applicable for in vivo purposes. Unlike other fabrication techniques, microgel characteristics can be modified by changing the starting polymer molecular weight. Increasing the starting PEG molecular weight increased microgel diameter and swelling ratio. Further modifications are suggested such as encapsulating molecules during microgel crosslinking. Simple adaptations to the PEG microgel building blocks are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Increasing the PEG molecular weight increased microgel diameter and swelling ratio. Simple adaptations to the PEG microgel precipitation reaction are explored for future applications of microgels as drug delivery vehicles and tissue engineering scaffolds.

This work describes the formation of poly(ethylene glycol) (PEG) microgels via a photopolymerized precipitation reaction. Precipitation reactions offer ...

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

아피시아 단일 뉴런의 전기 생리 및 분자 분석을 위한 신경리아 준비

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

Microscale Vortex-assisted Electroporator for Sequential Molecular Delivery

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant exogenous molecular probes into cells. This work reports a microfluidic electroporation platform capable of performing multiple molecule delivery to mammalian cells with precise and molecular-dependent parameter control. The system&#8217;s ability to isolate cells with uniform size distribution allows for less variation in electroporation efficiency per given electric field strength; hence enhanced sample viability. Moreover, its process visualization feature allows for observation of the fluorescent molecular uptake process in real-time, which permits prompt molecular delivery parameter adjustments in situ for efficiency enhancement. To show the vast capabilities of the reported platform, macromolecules with different sizes and electrical charges (e.g., Dextran with MW of 3,000 and 70,000 Da) were delivered to metastatic breast cancer cells with high delivery efficiencies (&#62;70%) for all tested molecules. The developed platform has proven its potential for use in the expansion of research fields where on-chip electroporation techniques can be beneficial.

A microfluidic vortex assisted electroporation platform was developed for sequential delivery of multiple molecules into identical cell populations with precise and independent dosage control. The system&#8217;s size based target cell purification step preceding electroporation aided to enhance molecular delivery efficiency and processed cell viability.

마이크로 소용돌이를 이용한 순차 분자 전달을위한 Electroporator을

Electroporation has received increasing attention in the past years, because it is a very powerful technique for physically introducing non-permeant ...

Biofunctionalized Prussian Blue Nanoparticles for Multimodal Molecular Imaging Applications

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, complementary imaging techniques. These imaging agents facilitate the real-time assessment of pathways and mechanisms in vivo, which enhance both diagnostic and therapeutic efficacy. This article presents the protocol for the synthesis of biofunctionalized Prussian blue nanoparticles (PB NPs) - a novel class of agents for use in multimodal, molecular imaging applications. The imaging modalities incorporated in the nanoparticles, fluorescence imaging and magnetic resonance imaging (MRI), have complementary features. The PB NPs possess a core-shell design where gadolinium and manganese ions incorporated within the interstitial spaces of the PB lattice generate MRI contrast, both in T1 and T2-weighted sequences. The PB NPs are coated with fluorescent avidin using electrostatic self-assembly, which enables fluorescence imaging. The avidin-coated nanoparticles are modified with biotinylated ligands that confer molecular targeting capabilities to the nanoparticles. The stability and toxicity of the nanoparticles are measured, as well as their MRI relaxivities. The multimodal, molecular imaging capabilities of these biofunctionalized PB NPs are then demonstrated by using them for fluorescence imaging and molecular MRI in vitro.

This protocol describes the synthesis of biofunctionalized Prussian blue nanoparticles and their use as multimodal, molecular imaging agents. The nanoparticles have a core-shell design where gadolinium or manganese ions within the nanoparticle core generate MRI contrast. The biofunctional shell contains fluorophores for fluorescence imaging and targeting ligands for molecular targeting.

멀티 모달 분자 이미징 애플리케이션을위한 Biofunctionalized 프 러시안 블루 나노 입자

Multimodal, molecular imaging allows the visualization of biological processes at cellular, subcellular, and molecular-level resolutions using multiple, ...

Engineering Molecular Recognition with Bio-mimetic Polymers on Single Walled Carbon Nanotubes

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the optical window where biological samples are most transparent. Here, we outline techniques to adsorb amphiphilic polymers and polynucleic acids onto the surface of SWNTs to engineer their corona phases and create novel molecular sensors for small molecules and proteins. These functionalized SWNT sensors are both biocompatible and stable. Polymers are adsorbed onto the nanotube surface either by direct sonication of SWNTs and polymer or by suspending SWNTs using a surfactant followed by dialysis with polymer. The fluorescence emission, stability, and response of these sensors to target analytes are confirmed using absorbance and near-infrared fluorescence spectroscopy. Furthermore, we demonstrate surface immobilization of the sensors onto glass slides to enable single-molecule fluorescence microscopy to characterize polymer adsorption and analyte binding kinetics.

We present a protocol for engineering the corona phase of near infrared fluorescent single walled carbon nanotubes (SWNTs) using amphiphilic polymers and DNA to develop sensors for molecular targets without known recognition elements.

단일 벽 탄소 나노 튜브에 바이오 모방 고분자와 공학 분자 인식

Semiconducting single-wall carbon nanotubes (SWNTs) are a class of optically active nanomaterial that fluoresce in the near infrared, coinciding with the ...

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and macro-structures, as seen in the cases of moving water in vascular bundles and moisturizing water in skin layers. In this study, we developed a method for assessing the effect of aqueous liquid crystalline (LC) solutions composed of biopolymers on drying. As LC biopolymers have megamolecular weight, we chose to study polysaccharides, cytoskeletal proteins, and DNA. The observation of biopolymer solutions during drying under polarized light reveals milliscale self-integration starting from the unstable air-LC interface. The dynamics of the aqueous LC biopolymer solutions can be monitored by evaporating water from a one-side-open cell. By analyzing the images taken using cross-polarized light, it is possible to recognize the spatio-temporal changes in the orientational order parameter. This method can be useful for the characterization of not only artificial materials in various fields, but also natural living tissues. We believe that it will provide an evaluation method for soft materials in the biomedical and environmental fields.

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.