نود أن نشكر مؤسسة فودافون الأمريكتين، ومؤسسة بيل وميليندا غيتس، ومؤسسة أبحاث ساو باولو (FAPESP) لتمويل هذا البحث. ونود أيضا أن نشكر العديد من أعضاء هيئة التدريس من قسم علم الحشرات في جامعة كاليفورنيا، ريفرسايد، لنصائحهم في هذا المشروع.

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	<li>Kahn, M. C., Celestin, W., Offenhauser, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recording+of+sounds+produced+by+certain+disease-carrying+mosquitoes.">Recording of sounds produced by certain disease-carrying mosquitoes.</a> Science. 101 (2622), 335-336 (1945).</li><li>Reed, S. C., Williams, C. M., Chadwick, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Frequency+of+wing-beat+as+a+character+for+separating+species+races+and+geographic+varieties+of+Drosophila.">Frequency of wing-beat as a character for separating species races and geographic varieties of Drosophila.</a> Genetics. 27 (3), 349 (1942).</li><li>Belton, P., Costello, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flight+sounds+of+the+females+of+some+mosquitoes+of+Western+Canada.">Flight sounds of the females of some mosquitoes of Western Canada.</a> Entomologia experimentalis et applicata. 26 (1), 105-114 (1979).</li><li>Mankin, R. W., Machan, R., Field Jones, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=testing+of+a+prototype+acoustic+device+for+detection+of+Mediterranean+fruit+flies+flying+into+a+trap.">testing of a prototype acoustic device for detection of Mediterranean fruit flies flying into a trap.</a> Proc. 7th Int Symp Fruit Flies of Economic Importance. , 10-15 (2006).</li><li>Raman, D. R., Gerhardt, R. R., Wilkerson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+insect+flight+sounds+in+the+field+Implications+for+acoustical+counting+of+mosquitoes.">Detecting insect flight sounds in the field Implications for acoustical counting of mosquitoes.</a> Transactions of the ASABE. 50 (4), 1481 .</li><li>Moore, A., Miller, J. R., Tabashnik, B. E., Gage, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+identification+of+flying+insects+by+analysis+of+wingbeat+frequencies.">Automated identification of flying insects by analysis of wingbeat frequencies.</a> Journal of economic entomology. 79 (6), 1703-1706 (1986).</li><li>Moore, A., Miller, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+identification+of+optically+sensed+aphid+(Homoptera:+Aphidae)+wingbeat+waveforms.">Automated identification of optically sensed aphid (Homoptera: Aphidae) wingbeat waveforms.</a> Annals of the Entomological Society of America. 95 (1), 1-8 (2002).</li><li>Li, Z., Zhou, Z., Shen, Z., Yao, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+identification+of+mosquito+(diptera:+Culicidae)+wingbeat+waveform+by+artificial+neural+network.">Automated identification of mosquito (diptera: Culicidae) wingbeat waveform by artificial neural network.</a> Artificial Intelligence Applications and Innovations. , 483-489 (2005).</li><li>Moore, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Artificial+neural+network+trained+to+identify+mosquitoes+in+flight.">Artificial neural network trained to identify mosquitoes in flight.</a> Journal of insect behavior. 4 (3), 391-396 (1991).</li><li>Halevy, A., Norvig, P., Pereira, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+unreasonable+effectiveness+of+data.">The unreasonable effectiveness of data.</a> Intelligent Systems, IEEE. 24 (2), 8-12 (2009).</li><li>Banko, M., Brill, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitigating+the+paucity-of-data+problem+exploring+the+effect+of+training+corpus+size+on+classifier+performance+for+natural+language+processing.">Mitigating the paucity-of-data problem exploring the effect of training corpus size on classifier performance for natural language processing.</a> , 1-5 (2001).</li><li>Shotton, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+human+pose+recognition+in+parts+from+single+depth+images.">Real-time human pose recognition in parts from single depth images.</a> Communications of the ACM. 56 (1), 116-124 (2013).</li><li>Ilyas, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Classifying+insects+on+the+fly.">Classifying insects on the fly.</a> Ecological Informatics. , (2013).</li><li>Mack, Y. P., Rosenblatt, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multivariate+k-nearest+neighbor+density+estimates.">Multivariate k-nearest neighbor density estimates.</a> Journal of Multivariate Analysis. 9 (1), 1-15 (1979).</li><li>Benedict, M., Robinson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+releases+of+transgenic+mosquitoes+an+argument+for+the+sterile+insect+technique.">The first releases of transgenic mosquitoes an argument for the sterile insect technique.</a> TRENDS in Parasitology. 19 (8), 349-355 (2003).</li><li>. Supporting Materials Available from: <a target="_blank" href="https://sites.google.com/site/insectclassification">https://sites.google.com/site/insectclassification</a></li><li>Chen, Y., Why, A., Batista, G., Mafra-Neto, A., Keogh, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flying+Insect+Classification+with+Inexpensive+Sensors.">Flying Insect Classification with Inexpensive Sensors.</a> Journal of Insect. 27 (5), 657-677 (2014).</li></ol>

تعلن الكتاب أنه ليس لديهم المصالح المالية المتنافسة.

الإطار الاستشعار / تصنيف الموصوفة هنا يسمح للتصنيف غير مكلفة وقابلة للمن الحشرات الطائرة. على دقة قابلة للتحقيق من قبل النظام هي جيدة بما فيه الكفاية للسماح تطوير المنتجات التجارية ويمكن أن تكون أداة مفيدة في البحث الحشري. إن الق...

فكرة تصنيف الحشرات تلقائيا باستخدام الصوت عرضية من رحلتهم يعود تاريخها إلى الأيام الأولى من أجهزة الكمبيوتر المتاحة تجاريا ومعدات تسجيل الصوت 1. ومع ذلك، فقد تم إحراز تقدم ضئيل على هذه المشكلة في العقود الفاصلة. ويمكن أن يعزى عدم إحراز تقدم في هذا السعي إلى عدة عوامل ذات الصلة. أولا، جعلت قلة من أجهزة الاستشعار فعالة لجمع البيانات صعبا. وقد استخدمت معظم الجهود لجمع البيانات الميكروفونات الصوتية 2-5. هذه الأجهزة هي حساسة للغاية لتصفية الضجيج والضوضاء المحيطة في البيئة، مما أدى إلى بيانات ضئيلة جدا وذات جودة منخفضة. الثانية، مما يضاعف هذه القضايا جودة البيانات هو حقيقة أن العديد من الباحثين قد حاولوا معرفة تصنيف نماذج معقدة للغاية، وخاصة الشبكات العصبية 6-8. محاولة لمعرفة نماذج تصنيف معقدة، مع مجرد عشرات الأمثلة،وصفة لالإفراط في تركيب. ثالثا، صعوبة الحصول على البيانات يعني أن حاول العديد من الباحثين على بناء نماذج تصنيف مع بيانات محدودة جدا، عدد قليل من الحالات 9 300 أو أقل. ومع ذلك، فمن المعروف أن لبناء نماذج تصنيف دقيقة، والمزيد من بيانات أفضل 10-13. ويتناول هذا العمل جميع القضايا الثلاث. البصرية (بدلا من الصوتية) يمكن استخدام أجهزة الاستشعار لتسجيل &quot;سليمة&quot; للطيران الحشرات من متر، مع الثابتية الكامل لتصفية الضوضاء والأصوات المحيطة. وقد سمحت هذه المجسات لتسجيل الملايين من حالات التدريب المسمى، أكثر بكثير من البيانات كل الجهود السابقة مجتمعة، وبالتالي يساعد على تجنب الجهود البحثية السابقة الإفراط المناسب التي يعاني منها. وهناك طريقة المبدئي هو مبين أدناه أن يسمح بإدراج معلومات إضافية في نموذج التصنيف. يمكن أن تكون هذه المعلومات الإضافية كما اليومية المعتادة كما وسهلة والحصول على الوقت من اليوم، ومع ذلك لا تزال تنتج مكاسب كبيرة في دقة النموذج. وأخيرا، أظهرت أن كميات هائلة من البيانات التي تم جمعها تسمح لنا للاستفادة من &quot;فاعلية غير معقولة من البيانات&quot; 10 لإنتاج المصنفات بسيطة ودقيقة وقوية. باختصار، انتقلت تحلق تصنيف الحشرات وراء مطالبات مشكوك فيها تم إنشاؤها في مختبر البحوث وجاهز الآن للنشر في العالم الحقيقي. وأجهزة الاستشعار والبرمجيات المقدمة في هذا العمل توفر أدوات قوية الباحثين في جميع أنحاء العالم لتسريع أبحاثهم.

<table border="0" cellpadding="0" cellspacing="0" width="1293">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Name of Material/ Equipment</td>
			<td style="width:237px;">Company</td>
			<td style="width:257px;">Catalog Number</td>
			<td style="width:471px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Audio Recorder: ICD-PX312</td>
			<td style="width:237px;">Sony</td>
			<td style="width:257px;">4-267-065-11(2)</td>
			<td>With a 8 GB microSD extra memory</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Insectary</td>
			<td style="width:237px;">Lee&#39;s Aquarium &#38; Pet Products.</td>
			<td style="width:257px;">20088 HerpHaven&#174;, Large Rectangle</td>
			<td style="width:471px;">14 1/2&#34; Long x 8 3/4&#34; Wide x 9 3/4&#34; high. Modified to house insects.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:328px;">Laser Line Generator, 650nm (red)</td>
			<td style="width:237px;">Apinex (www.apinex.com)</td>
			<td style="width:257px;">LN60-650</td>
			<td style="width:471px;">5mW. This is a low powered laser, similar to a teachers lasers pointer</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:328px;">Photodiode Array</td>
			<td style="width:237px;">VISHAY SEMICONDUCTOR&#160; TEFD4300&#160; PIN PHOTODIODE, 650NM, 20DEG, T1</td>
			<td style="width:257px;">TEFD4300</td>
			<td style="width:471px;">We made a custom array of 15 of these Photodiodes wired in parallel</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Analogue to Digital Convertor Integrated Circuit&#160;</td>
			<td style="width:237px;">Custom made in our lab</td>
			<td style="width:257px;"></td>
			<td style="width:471px;">We made this item ourselves, but an easily available commercial product, Gino PCF8591 AD/DA Converter, provides the same functionality.&#160;</td>
		</tr>
	</tbody>
</table>

flying insect detection and classification with inexpensive sensors

ومن شأن نظام موسع وغير مكلفة يمكن أن تصنف بدقة الحشرات الطائرة لها انعكاسات هامة على الأبحاث الحشري، والسماح لتطوير العديد من التطبيقات المفيدة في النواقل ومكافحة الآفات لكل من علم الحشرات الطبي والزراعي. ونظرا لهذا، شهدت السنوات الستين الماضية العديد من الجهود البحثية المكرسة لهذه المهمة. حتى الآن، ومع ذلك، كان لا شيء من هذا البحث تأثير دائم. في هذا العمل، وتبين لنا أن أجهزة الاستشعار البصرية الزائفة الصوتية يمكن أن تنتج بيانات متفوقة. أن ميزات إضافية، سواء جوهري وخارجي لسلوك رحلة الحشرة، يمكن استغلالها لتحسين تصنيف الحشرات. أن النهج تصنيف النظرية الافتراضية يتيح للتعلم بكفاءة تصنيف النماذج التي هي قوية جدا إلى أكثر من تركيب، وإطار التصنيف العام يسمح لدمج بسهولة التعسفي عدد من الميزات. نحن لشرح النتائج مع التجارب واسعة النطاق التي تقزم جميع الأعمال السابقة كومبيNED، كما يقاس بعدد من الحشرات وعدد الأنواع التي تعتبر.

يتم عرض تجربتين هنا. لكل من التجارب، والبيانات المستخدمة وتم أخذ عينات بشكل عشوائي من مجموعة البيانات التي تحتوي على أكثر من 100،000 الكائنات. وتظهر التجربة الأولى قدرة المصنف المقترحة لتصنيف الأنواع المختلفة بدقة / الجنسين من الحش...

Watch this Scientific Journal Video about Flying Insect Detection and Classification with Inexpensive Sensors at JoVE.com

Flying Insect Detection and Classification with Inexpensive Sensors

اقترحنا نظام يستخدم غير مكلفة، موسع أجهزة الاستشعار البصرية الزائفة الصوتية تلقائيا وبدقة لكشف والعد، وتصنيف الحشرات الطائرة على أساس الصوت سفرهم.

تحلق كشف وتصنيف الحشرات مع مجسات رخيصة

An inexpensive, noninvasive system that could accurately classify flying insects would have important implications for entomological research, and allow ...

flying-insect-detection-and-classification-with-inexpensive-sensors

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

25.0K Views.  University of California, Riverside.  اقترحنا نظام يستخدم غير مكلفة، موسع أجهزة الاستشعار البصرية الزائفة الصوتية تلقائيا وبدقة لكشف والعد، وتصنيف الحشرات الطائرة على أساس الصوت سفرهم. 

Video: Flying Insect Detection and Classification with Inexpensive Sensors

Fluorescence detection methods for microfluidic droplet platforms

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that accompany system miniaturisation. Advantages include the ability to efficiently process nano- to femoto- liter volumes of sample, facile integration of functional components, an intrinsic predisposition towards large-scale multiplexing, enhanced analytical throughput, improved control and reduced instrumental footprints.1
In recent years much interest has focussed on the development of droplet-based (or segmented flow) microfluidic systems and their potential as platforms in high-throughput experimentation.2-4 Here water-in-oil emulsions are made to spontaneously form in microfluidic channels as a result of capillary instabilities between the two immiscible phases. Importantly, microdroplets of precisely defined volumes and compositions can be generated at frequencies of several kHz. Furthermore, by encapsulating reagents of interest within isolated compartments separated by a continuous immiscible phase, both sample cross-talk and dispersion (diffusion- and Taylor-based) can be eliminated, which leads to minimal cross-contamination and the ability to time analytical processes with great accuracy. Additionally, since there is no contact between the contents of the droplets and the channel walls (which are wetted by the continuous phase) absorption and loss of reagents on the channel walls is prevented.
Once droplets of this kind have been generated and processed, it is necessary to extract the required analytical information. In this respect the detection method of choice should be rapid, provide high-sensitivity and low limits of detection, be applicable to a range of molecular species, be non-destructive and be able to be integrated with microfluidic devices in a facile manner. To address this need we have developed a suite of experimental tools and protocols that enable the extraction of large amounts of photophysical information from small-volume environments, and are applicable to the analysis of a wide range of physical, chemical and biological parameters. Herein two examples of these methods are presented and applied to the detection of single cells and the mapping of mixing processes inside picoliter-volume droplets. We report the entire experimental process including microfluidic chip fabrication, the optical setup and the process of droplet generation and detection.

Droplet-based microfluidic platforms are promising candidates for high throughput experimentation since they are able to generate picoliter, self-compartmentalized vessels inexpensively at kHz rates. Through integration with fast, sensitive and high resolution fluorescence spectroscopic methods, the large amounts of information generated within these systems can be efficiently extracted, harnessed and utilized.

طرق الكشف عن منصات مضان الحبرية ميكروفلويديك

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed secondary tumors1,2,3. CTCs are indicators of metastatic disease and their detection in blood samples may be used to diagnose cancer and monitor a patient&prime;s response to therapy. Since CTCs are rare, comprising about one tumor cell among billions of normal blood cells in advanced cancer patients, their detection and enumeration is a difficult task. We exploit the presence of pigment in most melanoma cells to generate photoacoustic, or laser induced ultrasonic waves in a custom flow cytometer for detection of circulating melanoma cells (CMCs)4,5. This process entails separating a whole blood sample using centrifugation and obtaining the white blood cell layer. If present in whole blood, CMCs will separate with the white blood cells due to similar density. These cells are resuspended in phosphate buffered saline (PBS) and introduced into the flowmeter. Rather than a continuous flow of the blood cell suspension, we induced two phase flow in order to capture these cells for further study. In two phase flow, two immiscible liquids in a microfluidic system meet at a junction and form alternating slugs of liquid6,7. PBS suspended white blood cells and air form microliter slugs that are sequentially irradiated with laser light. The addition of a surfactant to the liquid phase allows uniform slug formation and the user can create different sized slugs by altering the flow rates of the two phases. Slugs of air and slugs of PBS with white blood cells contain no light absorbers and hence, do not produce photoacoustic waves. However, slugs of white blood cells that contain even single CMCs absorb laser light and produce high frequency acoustic waves. These slugs that generate photoacoustic waves are sequestered and collected for cytochemical staining for verification of CMCs.

We have developed a flow cytometer using laser induced ultrasound to detect circulating melanoma cells as an early indicator of metastatic disease.

كشف وعزل خلايا الميلانوما تعميم استخدام Flowmetry الضوئي

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

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 ...

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

جهاز الاستشعار البيولوجي للكشف عن المضادات الحيوية البكتيريا العنقودية الذهبية المقاومة لل

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Glutamine Flux Imaging Using Genetically Encoded Sensors

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for biological molecules became available in the past 15 years, some of which became indispensable tools that are used routinely in many laboratories.
One of the exciting applications of genetically encoded sensors is the use of these sensors in investigating cellular transport processes. Properties of transporters such as kinetics and substrate specificities can be investigated at a cellular level, providing possibilities for cell-type specific analyses of transport activities. In this article, we will demonstrate how transporter dynamics can be observed using genetically encoded glutamine sensor as an example. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed.

This article will demonstrate how to monitor glutamine dynamics in live cells using FRET. Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. Experimental design, technical details of the experimental settings, and considerations for post-experimental analyses will be discussed for genetically encoded glutamine sensors.

الجلوتامين الجريان التصوير باستخدام مجسات مشفرة وراثيا

Genetically encoded sensors allow real-time monitoring of biological molecules at a subcellular resolution. A tremendous variety of such sensors for ...

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.

وبيوشيب الكهروكيميائية استنادا ميكروفلويديك لتسمية خالية تحليل الحمض النووي التهجين

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

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of exosomes to determine if they carry disease discriminatory biomarkers. For performing exosomal analysis, it is necessary to develop a method for extracting and analyzing exosomes from target biofluids without damaging the internal content.
Electric field-induced release and measurement (EFIRM) is a method for specifically extracting exosomes from biofluids, unloading their cargo, and testing their internal RNA/protein content. Using an anti-human CD63 specific antibody magnetic microparticle, exosomes are first precipitated from biofluids. Following extraction, low-voltage electric cyclic square waves (CSW) are applied to disrupt the vesicular membrane and cause cargo unloading. The content of the exosome is hybridized to DNA primers or antibodies immobilized on an electrode surface for quantification of molecular content.
The EFIRM method is advantageous for extraction of exosomes and unloading cargo for analysis without lysis buffer. This method is capable of performing specific detection of both RNA and protein biomarker targets in the exosome. EFIRM extracts exosomes specifically based on their surface markers as opposed to size-based techniques.
Transmission electron microscopy (TEM) and assay demonstrate the functionality of the method for exosome capture and analysis. The EFIRM method was applied to exosomal analysis of 9 mice injected with human lung cancer H640 cells (a cell line transfected to express the exosome marker human CD63-GFP) in order to test their exosome profile against 11 mice receiving saline controls. Elevated levels of exosomal biomarkers (reference gene GAPDH and protein surface marker human CD63-GFP) were found for the H640 injected mice in both serum and saliva samples. Furthermore, saliva and serum samples were demonstrated to have linearity (R = 0.79). These results are suggestive for the viability of salivary exosome biomarkers for detection of distal diseases.

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM

Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves.

الكشف عن Exosomal العلامات البيولوجية التي يسببها الميدانية الكهربائية الإصدار والقياس (EFIRM)

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of ...

Label-free Single Molecule Detection Using Microtoroid Optical Resonators

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental studies on the behavior of molecules. Single molecule detection techniques commonly utilize labels such as fluorescent tags or quantum dots, however, labels are not always available, increase cost and complexity, and can perturb the events being studied. Optical resonators have emerged as a promising means to detect single molecules without the use of labels. Currently the smallest particle detected by a non-plasmonically-enhanced bare optical resonator system in solution is a 25 nm polystyrene sphere1. We have developed a technique known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that can surpass this limit and achieve label-free single molecule detection in aqueous solution2. As signal strength scales with particle volume, our work represents a &#62; 100x improvement in the signal to noise ratio (SNR) over the current state of the art. Here the procedures behind FLOWER are presented in an effort to increase its usage in the field.

We have developed a label-free biosensing system based on optical resonator technology known as Frequency Locking Optical Whispering Evanescent Resonator (FLOWER) that is capable of detecting single molecules in solution. Here the procedures behind this work are described and presented.

تسمية خالية واحدة كشف الجزيئة عن طريق Microtoroid البصرية الرنين

Detecting small concentrations of molecules down to the single molecule limit has impact on areas such as early detection of disease, and fundamental ...

Sensitive Detection of Proteopathic Seeding Activity with FRET Flow Cytometry

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and progression of pathology in several neurodegenerative diseases, including Alzheimer's disease and the related tauopathies. The potentially critical role of tau seeds in disease progression strongly supports the need for a sensitive assay that readily detects seeding activity in biological samples. 
By combining the specificity of fluorescence resonance energy transfer (FRET), the sensitivity of flow cytometry, and the stability of a monoclonal cell line, an ultra-sensitive seeding assay has been engineered and is compatible with seed detection from recombinant or biological samples, including human and mouse brain homogenates. The assay employs monoclonal HEK 293T cells that stably express the aggregation-prone repeat domain (RD) of tau harboring the disease-associated P301S mutation fused to either CFP or YFP, which produce a FRET signal upon protein aggregation. The uptake of proteopathic tau seeds (but not other proteins) into the biosensor cells stimulates aggregation of RD-CFP and RD-YFP, and flow cytometry sensitively and quantitatively monitors this aggregation-induced FRET. The assay detects femtomolar concentrations (monomer equivalent) of recombinant tau seeds, has a dynamic range spanning three orders of magnitude, and is compatible with brain homogenates from tauopathy transgenic mice and human tauopathy subjects. With slight modifications, the assay can also detect seeding activity of other proteopathic seeds, such as &#945;-synuclein, and is also compatible with primary neuronal cultures. The ease, sensitivity, and broad applicability of FRET flow cytometry makes it useful to study a wide range of protein aggregation disorders.

Cell-to-cell transfer of protein aggregates, or proteopathic seeds, may underlie the progression of pathology in neurodegenerative diseases. Here, a novel FRET flow cytometry assay is described that enables specific and sensitive detection of seeding activity from recombinant or biological samples.

كشف حساسية Proteopathic البذر آخر مع الحنق التدفق الخلوي

Increasing evidence supports transcellular propagation of toxic protein aggregates, or proteopathic seeds, as a mechanism for the initiation and ...

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW &#60; 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 &#186;C. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

سريع المعالجة الأنزيمية من البروتينات للكشف عن مرض التصلب العصبي المتعدد مع Microreactor التدفق من خلال

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...