Sandia Birleşik Sözleşme DE-AC04-94AL85000 altında Enerji Ulusal Nükleer Güvenlik İdaresi Bölümü Belirtilen için Sandia Corporation, Lockheed Martin Şirketi tarafından işletilen MultiProgram laboratuvardır. Yazarlar Sandia Ulusal Laboratuvarı &#39;Laboratuar fon kabul Araştırma ve Geliştirme (LDRD) programı Directed.

1. Microneedle Fabrikasyon <ol><li> Üç boyutlu modelleme yazılımı Solidworks (Dassault Systemes SA, Velizy, Fransa) kullanarak, bir piramit şeklindeki içi boş microneedle dizisi tasarlamak (Şekil 1). 3-5 </li><li> Magics RP 13 yazılım (Materialise NV, Leuven, Belçika) kullanarak microneedle dizi için bir destek yapısı tasarlayın. Destek yapısı reçine imalat sırasında cihaz akmasına izin veriyor ve microneedles dayandığı bir temel sağlar. Örneğin, bir destek yapısı Şekil 1 &#39;de gösterilmiştir. </li><li> Bağlantılı destek ve microneedle dizi dosyaları üretim süreci kontrol Perfactory RP yazılımı (EnvisionTEC GmbH, Gladbeck, Almanya), içine yüklenir. Bu yazılım paketi içinde, fabrikasyon olarak microneedle dizilerin sayısını seçin ve fabrikasyon plaka üzerinde cihazların yerleşimini belirleyecektir. </li><li> Perfactory hızlı pr için 180 mW de ultraviyole modunda çalıştırın kalibrasyonototyping üretim sistemi ve enerji sapma doğrulamak ± 2 mW içinde. </li><li> Imalat tamamlandıktan sonra, 15 dakika isopropanole tablaya gelen microneedle diziler kaldırmak ve geliştirmek. Sıkıştırılmış hava ile dizileri tamamen kuruması ve polimerizasyon sağlamak için Otoflash Postcuring Sistemi (EnvisionTEC GmbH, Gladbeck, Almanya) içinde 50 saniye süreyle, oda sıcaklığında microneedles tedavisi. </li><li> Mikroskobu ile microneedle fabrikasyon Validate ve her microneedle delik içi boş ve engelsiz olduğunu doğrulayın. Tam-imal microneedles Şekil 2 &#39;de gösterilmiştir. </li></ol> 2. Karbon Yapıştır Elektrot Diziler Fabrikasyon <ol><li> Delikler kesip oldu düz esnek kablo (21039-0249), altta yatan bireysel adreslenebilir bağlayan bakır teller ortaya çıkarmak için bir 60 W Model 6.75 CO 2 ve raster / vektör lazer sistemi (Universal Laser Systems, Inc, Scottsdale, AZ) kullanın ticari bir kaynağı (Mo elde edilenlex Bağlayıcı Corp, Lisle, IL) (Şekil 3 (A ve B)). Uygun lazer ablasyon tabağa hizalamak için bir jig içinde düz esnek kablolar yerleştirin. Esnek bir kablo yalıtım kısmında 500 um çapındaki oyukları oluşturmak için bir yaklaşım rastering kullanabilir. Ablasyon için Patterns CorelDraw Oluşturuldu (Corel, Ottawa, Ontario) ve lazer sistemine gönderilir. </li><li> 40 psi bir airbrush bu spreyler aseton ile değiştirilmiş yassı esnek kablolar temizleyin. Izopropanol ve deiyonize su ile durulayarak bunları temizleme tamamlayın. Hiçbir yalıtım filmi maruz bakır şeritler üzerinde kaldığı bir mikroskop altında doğrulayın. </li><li> Bir sonraki adım karbon macunlar ambalajlanması için bir holding boşluğu yaratmaktır. Melinex bandı (basınca duyarlı akrilik yapıştırıcı ile tek bir tarafı kaplanmıştır 0.002 &quot;kalınlığında) elektrot ablasyon elektrot şeritler üzerine odaklı şeritler, ve düzgün bir bağlantı sağlamak için 2 dakika için 3000 psi sıkıştırılmış olarak aynı deseni ile ablate edilmektir. Bu case, kavite çapı 750 mikron olduğunu. </li><li> Melinex bant ek bir katman (0.004 &quot;basınca duyarlı akrilik yapıştırıcı ile iki tarafı kaplı kalınlığı) daha sonra tek taraflı yapışkan bant ile aynı desen ablaze ve karbon pasta elektrot dizileri için tahvil microneedle diziler uyumu sonra kullanılır . </li></ol> 3. Fonksiyonel Karbon Pastalar Sentezi ve Elektrot Çürükler ve Paketleme <ol><li> Glükoz duyarlı karbon pasta önceki bir tarifi off dayanır ve homojen bir karışım elde edilinceye kadar glukoz oksidaz ve poli (ethylenimine) 2.2 mg 10 mg karıştırılması ile elde edilir. 16 bu karışım, karbon üzerinde rodyum tozu, 60 mg (için % 5 yükleme) ilave edilir. 40 mg mineral yağ ilave edilir ve daha sonra karıştırılır. Macunlar hazırlandıktan sonra bir hafta kadar kullanılır; macunlar ° C&#39;de kullanılıncaya kadar 4 depolanır. </li><li> PH duyarlı karbon pasta karışım% 30 (w / w), mineral yağ ve% 70 (w / w) grafit p ile elde edilirowder. Bölüm 3.4 &#39;de açıklandığı gibi elektrot boşluğuna yapıştırın paketleyin. 0,5 M fosforik asit içinde 10 mM Fast Blue RR diazonyum tuzu (4-benzoilamino-2 ,5-dimethoxybenzenediazonium klorür hemi (çinko klorür) tuz) çözeltisi. 17 yapmak için, paketlenmiş macun elektrot üzerinde bu çözelti, 20 ul damla yerleştirin 30 dakika kendiliğinden Fast Blue PR diazonyum tuzu chemisorb için. Tampon veya deiyonize su kullanılmadığı da deiyonize su ve mağaza ile durulayın. </li><li> Laktat duyarlı karbon pasta önceki bir tarifi off dayanır ve karbon tozu ve laktat oksidaz 2.5 mg üzerinde rodyum 2.5 mg karıştırılması ile elde edilir, beş rotasyon için sonikasyon ile 5 dakika ve vorteks ile 5 dakika arasında değişen. 18 </li><li> Hazırlanan düz esnek bir kablo içine modifiye macunlar ambalaj elektrot boşlukları üzerine ilgili macunlar uygulanarak gerçekleştirilir. Mala ve paketi t gibi plastik ince bir parçası (örneğin, plastik bir tartın teknenin bir kenarına) kullanmadüzgün bir yüzey elde edilene kadar o yapıştırın. Fazla macun çıkana kadar ikinci bir temiz ağırlığındaki tekne ile tekrarlayın. Deiyonize su ile yıkayın. Şematik karbon macunlar ambalaj, boşluklar oluşturmak için lazer ablasyon gösteren ve microneedle entegrasyon (bölüm 2 ve 3 açıklanan) Şekil 3&#39;te sunulmuştur. </li></ol> 4. Algılama ve sensör kalibrasyon <ol><li> Laktat algılama M fosfat tampon maddesi (pH = 7,5). Şekil 4 (a) laktat saptanması için elektrokatalitik reaksiyon şematik bir içerir -0,15 V sensörünün chronoamperometric tepkisi ölçerek ve 0.1 &#39;de 15 saniye sonra, mevcut kayıt gerçekleştirilir . </li><li> Glikoz algılama -0,05 V sensörünün chronoamperometric tepkisi ölçerek ve 0.1 M fosfat tampon maddesi (pH 7.0) içinde 15 saniye sonra, mevcut kayıt ile benzer bir şekilde gerçekleştirilir. Şekil 4 (b) için elektrokatalitik reaksiyon şematik bir içerir dglikoz etection. </li><li> pH 100 mV / s -0.7 V 0.8 V siklik voltametrik taramaları çalıştıran ve oksidatif pik potansiyeli konumu kaydederek izlenir. PH tespiti için redoks reaksiyon şematik Şekil 5 de gösterilmiştir. </li><li> Glikoz ve laktat sensörleri için kalibrasyon eğrileri ilgili analit arda eklemelerle oluşturulabilir; bölümler 5.1 ve 5.2 &#39;de açıklandığı gibi chronoamperometric ölçümleri her analit eklenmesinden sonra yapmaktadır. Alternatif olarak, sabit bir potansiyel chronoamperometric ölçümleri mevcut stabilizasyonu için her bir analit yanı arasında yeterli bir süre (saniye ~ 10-100) izin verirken karıştırma altında yapılabilir. </li><li> pH kalibrasyon eğrileri 1.0 pH birimi artışlarla 5 ile 8 arasında bilinen bir pH değerleri serisi içinde oksidatif pik potansiyelinin ölçümü pozisyonu ve bölüm 5.3 &#39;te tarif edildiği gibi siklik voltamogramları kayıt tarafından oluşturulabilir. </li></ol> 5.. Temsilcisi Results Modifiye karbon yapıştırın dolu microneedles ile sakin çözümleri chronoamperometric eğrileri (örneğin, glukoz algılama veya laktat tespiti için) almak, geçerli derhal ilgili algılama potansiyeli başvurusu üzerine düşecektir. Sonunda kararlı bir duruma değerine azalacağı. Bir temsili Sonuç, Şekil 6&#39;da gösterilmiştir; bu sonuç, laktat microneedle az laktat ve kaydın 2 mM ilave elde edildi. Kısaca, her solüsyon laktat eklenmesinden sonra karıştırılmıştır edilmelidir. 15 saniyeden sonra mevcut laktat konsantrasyonu arttıkça artar üzerine; mevcut tepki sonra bilinmeyen bir çözelti içinde laktat konsantrasyonunu belirlemek için kullanılabilir. Alternatif olarak, sürekli izleme artan bir glukoz konsantrasyonu (Şekil 5) içeren bir çözelti için gösterildiği gibi karıştırılan bir çözeltisine (ya da bir akıcı solüsyonu içinde) da kullanılabilir. Yine, mevcut üzerinde artış t artanO glukoz konsantrasyonu bilinmeyen bir çözeltisine glikoz tepkisi standardize etmek için kullanılabilir. Yeterli zaman çözüm stabilize etmek için izin vermek için her başak sonra izin verilmelidir. 0.1 M fosfat tampon içinde pH duyarlı microneedle az siklik voltamogramları 5 ila Şekil 6 &#39;da 8 1 pH birimi artışlarla, dört farklı pH çözümler üzerinde gösterilir. PH artışı ile oksidatif pik potansiyeli kaymalar, bu fenomen pH değeri bir göstergesi olarak kullanılmıştır. <img alt="Şekil 1" src="/files/ftp_upload/4067/4067fig1.jpg" /> Şekil 1. Solidworks (A) ve destek yapısı (B) gösterir yazdırma ekranı, hazırlandı microneedle dizi STL dosyası görüntüleri. <img alt="Şekil 2" src="/files/ftp_upload/4067/4067fig2.jpg" /> Şekil 2. Microneedle dizi (A) ve bu dizi içinde tek microneedle (B) elektron mikroskop fotoğrafları tarama. Şekil 3. Düz esnek kablo takımı şematik. Söz adımları, düz esnek bir kablo (A) değiştirerek (B) desenli çevreler ablasyonu, karbon macun (C) ile doldurulur, ayrıca ikinci bir kesilip alınmış Melinex tabakası ekleyerek ve çiftleşme edilir başlangıçta ablazeydi Melinex tabakası, ilave içerir microneedle dizi (D). <a href="http://www.jove.com/files/ftp_upload/4067/4067fig3large.jpg" target="_blank">büyük rakam görmek için buraya tıklayın</a> . <img alt="Şekil 4" src="/files/ftp_upload/4067/4067fig4.jpg" /> Şekil 4. 0.1 M fosfat tamponu (pH = 7.5) -0.15 V az 15 saniye chronoamperometric taramaları ile laktat duyarlı macun Kalibrasyonu. Laktat, bir 2 mM ek olarak mevcut tekabül her artış. <img alt="Şekil 5," src="/files/ftp_upload/4067/4067fig5.jpg" /> Şekil 5. </sHer bir düşüş karşılık olarak bir glükoz 2 mM Ayrıca tekabül -0,05 V. azından chronoamperometric taramalar çalışan glukoz-hassas bir macun&gt; Kalibrasyon Trong. 0.1 M fosfat tampon solüsyonu (pH = 7,0) kalibrasyon boyunca karıştırıldı. Harici Ag / AgCİ referans ve Pt ve karşı elektrotlar Bu çalışmada kullanılan edildi. <img alt="Şekil 6" src="/files/ftp_upload/4067/4067fig6.jpg" /> PH duyarlı karbon Şekil 6. Döngüsel voltamogram (CV) 1 pH birimi artışlarla (çamurcun = pH 8.0, yeşil pH = 7.0, mor pH = 6.0, kırmızı pH = 5.0), pH 5-8 üzerinde 0.1 M fosfat tamponunda yapıştırın. Beşinci CV Ag / AgCİ referans ve Pt tel karşı elektrotlar karşılık analizi için kullanıldı.

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Çıkar çatışması ilan etti.

Bu microneedle bazlı sensörünün tasarım birden fazla cihaz yönlerine üretim öncesi olarak kabul edildi. Gerçek zamanlı olarak saptanması için, bu sensor kullanmak için, algılayıcının tepki süresi düşük olmalıdır; Bu protokolde, her bir test sensörü beş saniyeden altında bir tepki süresi sergiledi. Bu protokolde kullanılan macunlar da elektrot cevabı etkileyebilir elektroaktif biyomoleküllerin içeren vivo ortamlarda içinde seçicilik için seçilmiştir. Bileşimi yapıştırmak ...

<table border="1"><tbody><tr><td> Reaktif Adı </td><td> Şirket </td><td> Katalog numarası </td></tr><tr><td> Düz esnek kablo </td><td> Molex </td><td> 3302/10SF </td></tr><tr><td> 0.003 &quot;Side taraflı bant </td><td> Melinex </td><td></td></tr><tr><td> 0.004 &quot;Çift taraflı bant </td><td> Melinex </td><td></td></tr><tr><td> Laktat oksidaz </td><td> Sigma </td><td> L0638 </td></tr><tr><td> Glukoz oksidaz </td><td> Sigma </td><td> G7141 </td></tr><tr><td> Karbon üzerinde rodyum </td><td> Sigma </td><td> 206164 </td></tr><tr><td> Grafit tozu </td><td> Sigma </td><td> 385031000 </td></tr><tr><td> poli (ethylenimine) </td><td> Acros </td><td> 178570010 </td></tr><tr><td> Madeni yağ </td><td> Sigma </td><td> M5904 </td></tr><tr><td> Glikoz </td><td> Sigma &lt;/ Td&gt; <td> G8270 </td></tr><tr><td> Laktat </td><td> Sigma </td><td> L1750 </td></tr><tr><td> Fast Blue RR tuz </td><td> Sigma </td><td> F0500 </td></tr><tr><td> e-Shell 300 </td><td> EnvisionTEC </td><td></td></tr><tr><td> e-Shell 200 </td><td> EnvisionTEC </td><td></td></tr><tr><td> Ag / AgCİ referans elektrotuna </td><td> Basi </td><td> MF-2052 </td></tr><tr><td> Pt tel </td><td> Basi </td><td></td></tr><tr><td> PGSTAT12 AutolabPotentiostat </td><td> EcoChemie </td><td></td></tr><tr><td> Perfactory RP </td><td> EnvisionTEC </td><td></td></tr><tr><td> Ottoflash Postcuring sistemi </td><td> EnvisionTEC </td><td></td></tr><tr><td> Fosforik asit </td><td> Balıkçı </td><td> A366-4 </td></tr><tr><td> 60W Model 6.75 CO 2 ve raster / vektör lazer sistemi </td><td> Universal Lazer Sistemleri </td><td> PLS6.75 </td></tr><tr><td> CorelDraw&#39;a </td><td> Corel </td><td></td></tr><tr><td> Solidworks </td><td> Dassault Systemes </td><td> 2009 </td></tr><tr><td> Magics RP13 </td><td> Materialise </td><td></td></tr></tbody></table>

hollow microneedle-based sensor for multiplexed transdermal electrochemical sensing

Biyolojik-ilgili moleküllerin hızlı analiz için minimal invaziv çoğaltılmış izleme sisteminin geliştirilmesi kronik tıbbi durumlar onların ani fizyolojik devletin uyduruk değerlendirmesi muzdarip bireyler sunabilir. Ayrıca, karmaşık ve çok etkenli tıbbi koşulların analizi için bir araştırma aracı olarak hizmet verebilir. Gerçekleştirilecek böyle bir Çoklu Analit sensör amacıyla, kullanıcıya ağrı veya zarar vermeden gerçekleşmesi gerekir interstisyel sıvı örneklenmesi, minimal invaziv olmalı ve analiz hızlı yanı sıra seçici olmalıdır. Başlangıçta ağrısız ilaç dağıtımı için geliştirilmiş, microneedles deri yoluyla aşı ve farmakolojik ajanlar (örn., insülin) teslim etmek için kullanılmıştır. 1-2 bu cihazlar interstisyel alanına erişmeye beri mikroelektrotla entegre microneedles transdermal olarak kullanılabilir elektrokimyasal sensör. Glukoz, glutamat, laktat seçici algılama, hydrogen peroksit, ve askorbik asit transdüklenmesinden elemanı olarak hizmet veren karbon fiberler, karbon modifiye macunlar, ve platin kaplı polimer microneedles ile entegre microneedle-elektrot cihazlar ile ortaya konmuştur. 3-7,8 Bu microneedle sensör teknolojisi, birden fazla analitlerin yerinde ve eş zamanlı tespiti için yeni ve sofistike analitik yaklaşım sağlamıştır. Multiplexing hızlı ve minimal invazif bir şekilde karakterize etmek aksi zor karmaşık mikroçevrelerde, izleme imkanı sunuyor. Örneğin, bu teknolojinin hücre dışı düzeylerinde eş zamanlı olarak izlenmesi için yararlanılabilir, glukoz, laktat ve pH, 9 önemli metabolik hastalıkların 7,10-14 göstergeleri (örn., kanser proliferasyonu) ve egzersiz indüklenen asidozu olan. 15

Watch this Scientific Journal Video about Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing at JoVE.com

Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

Bu makale ayrıntıları Multiplex microneedle tabanlı sensör yapımı. Aygıt bir hızlı ve seçici bir şekilde birden fazla analitlerin in situ numune alma ve elektrokimyasal analizi için geliştirilmektedir. Biz klinik tıp tasavvur ve biyomedikal araştırmalar, bu microneedle tabanlı sensörler için kullanır.

Multiplexed Transdermal Elektrokimyasal Algılama Hollow microneedle tabanlı Sensör

The development of a minimally invasive multiplexed monitoring system for rapid analysis of biologically-relevant molecules could offer individuals ...

hollow-microneedle-based-sensor-for-multiplexed-transdermal

Research

JoVE Journal

Bioengineering

14.2K Görüntüleme.  University of North Carolina and North Carolina State University. Bu makale ayrıntıları Multiplex microneedle tabanlı sensör yapımı. Aygıt bir hızlı ve seçici bir şekilde birden fazla analitlerin in situ numune alma ve elektrokimyasal analizi için geliştirilmektedir. Biz klinik tıp tasavvur ve biyomedikal araştırmalar, bu microneedle tabanlı sensörler için kullanır.

Video: Hollow Microneedle-based Sensor for Multiplexed Transdermal Electrochemical Sensing

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

Biyomoleküler Algılama istihdam İnterferometrik Yansıtma Görüntüleme Sensörü (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Biyomühendislik

Multiplexed Single-molecule Force Proteolysis Measurements Using Magnetic Tweezers

The generation and detection of mechanical forces is a ubiquitous aspect of cell physiology, with direct relevance to cancer metastasis1, atherogenesis2 and wound healing3. In each of these examples, cells both exert force on their surroundings and simultaneously enzymatically remodel the extracellular matrix (ECM). The effect of forces on ECM has thus become an area of considerable interest due to its likely biological and medical importance4-7.
	Single molecule techniques such as optical trapping8, atomic force microscopy9, and magnetic tweezers10,11 allow researchers to probe the function of enzymes at a molecular level by exerting forces on individual proteins. Of these techniques, magnetic tweezers (MT) are notable for their low cost and high throughput. MT exert forces in the range of ~1-100 pN and can provide millisecond temporal resolution, qualities that are well matched to the study of enzyme mechanism at the single-molecule level12. Here we report a highly parallelizable MT assay to study the effect of force on the proteolysis of single protein molecules. We present the specific example of the proteolysis of a trimeric collagen peptide by matrix metalloproteinase 1 (MMP-1); however, this assay can be easily adapted to study other substrates and proteases.

In this article we describe the use of magnetic tweezers to study the effect of force on enzymatic proteolysis at the single molecule level in a highly parallelizable manner.

Manyetik cımbız kullanarak Multiplexed Tek molekül Kuvvetleri Proteoliz Ölçümleri

The  generation and detection of mechanical forces is a ubiquitous aspect of cell physiology,  with direct relevance to cancer metastasis1, atherogenesis2 ...

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.

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.

Elektrokimyasal Sensörler kullanarak Bakteriyel Algılama ve Tanıma

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

Sensing of Barrier Tissue Disruption with an Organic Electrochemical Transistor

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the passage of necessary ions and molecules. A breach in this barrier can be caused by a reduction in the extracellular calcium concentration. This reduction in calcium concentration causes a conformational change in proteins involved in the sealing of the barrier, leading to an increase of the paracellular flux. To mimic this effect the calcium chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N&#39;,N&#39;-tetra acetic acid (EGTA) was used on a monolayer of cells known to be representative of the gastrointestinal tract. Different methods to detect the disruption of the barrier tissue already exist, such as immunofluorescence and&#160;permeability assays. However, these methods are time-consuming and costly and not suited to dynamic or high-throughput measurements. Electronic methods for measuring barrier tissue integrity also exist for measurement of the transepithelial resistance (TER), however these are often costly and complex. The development of rapid, cheap, and sensitive methods is urgently needed as the integrity of barrier tissue is a key parameter in drug discovery and pathogen/toxin diagnostics. The organic electrochemical transistor (OECT) integrated with barrier tissue forming cells has been shown as a new device capable of dynamically monitoring barrier tissue integrity. The device is able to measure minute variations in ionic flux with unprecedented temporal resolution and sensitivity, in real time, as an indicator of barrier tissue integrity. This new method is based on a simple device that can be compatible with high throughput screening applications and fabricated at low cost.

The Organic Electrochemical Transistor is integrated with live cells and used to monitor ion flux across the gastrointestinal epithelial barrier. In this study, an increase in ion flux, related to disruption of tight junctions, induced by the presence of the calcium chelator EGTA (ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetra acetic acid), is measured.

Organik Elektrokimyasal Transistör Bariyer Doku bozulması ve Algılama

The gastrointestinal tract is an example of barrier tissue that provides a physical barrier against entry of pathogens and toxins, while allowing the ...

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.

Etiket-serbest DNA Hibridizasyon Analiz için mikroakışkan tabanlı Elektrokimyasal Biochip

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

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

Solvent destekli çift katlı lipid (SALB) Yöntemi ile biyomembran Fabrikasyon

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

için içi boş fiber Biyoreaktörler<em&gt; İn Vivo</em&gt; Benzeri Memeli Doku Kültürü

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Extraction of Plant-based Capsules for Microencapsulation Applications

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin exine capsules (SECs) are obtained when spores or pollen are processed so as to remove the internal sporoplasmic contents. The resulting hollow microcapsules exhibit a high degree of micromeritic uniformity and retain intricate microstructural features related to the particular plant species. Herein, we demonstrate a streamlined process for the production of SECs from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs. The current SEC isolation procedure has been recently optimized to significantly reduce the processing requirements which are conventionally used in SEC isolation, and to ensure the production of intact microcapsules. Natural L. clavatum spores are defatted with acetone, treated with phosphoric acid, and extensively washed to remove sporoplasmic contents. After acetone defatting, a single processing step using 85% phosphoric acid has been shown to remove all sporoplasmic contents. By limiting the acid processing time to 30 hr, it is possible to isolate clean SECs and avoid SEC fracturing, which has been shown to occur with prolonged processing time. Extensive washing with water, dilute acids, dilute bases, and solvents ensures that all sporoplasmic material and chemical residues are adequately removed. The vacuum loading technique is utilized to load a model protein (Bovine Serum Albumin) as a representative hydrophilic compound. Vacuum loading provides a simple technique to load various compounds without the need for harsh solvents or undesirable chemicals which are often required in other microencapsulation protocols. Based on these isolation and loading protocols, SECs provide a promising material for use in a diverse range of microencapsulation applications, such as, therapeutics, foods, cosmetics, and personal care products.

This protocol/manuscript describes a streamlined process for the production of sporopollenin exine capsules (SECs) from Lycopodium clavatum spores and for the loading of hydrophilic compounds into these SECs.

Mikroenkapsülasyon Uygulamaları için Ekstraksiyon Tesisi tabanlı Kapsüller

Microcapsules derived from plant-based spores or pollen provide a robust platform for a diverse range of microencapsulation applications. Sporopollenin ...

Snap Chip for Cross-reactivity-free and Spotter-free Multiplexed Sandwich Immunoassays

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands of micro-scale immunoassays performed simultaneously on a single chip. Sandwich assay format improves assay specificity by detecting each target with two antibodies, but suffers from cross-reactivity between reagents thus limiting their multiplexing capabilities. Antibody colocalization microarray (ACM) has been developed for cross-reactivity-free multiplexed protein detection, but requires an expensive spotter on-site for microarray fabrication during assays. In this work, we demonstrate a snap chip technology that transfers reagent from microarray-to-microarray by simply snapping two chips together, thus no spotter is needed during the sample incubation and subsequent application of detection antibodies (dAbs) upon storage of pre-spotted slides, dissociating the slide preparation from assay execution. Both single and double transfer methods are presented to achieve accurate alignment between the two microarrays and the slide fabrication for both methods are described. Results show that &#60;40 &#956;m alignment has been achieved with double transfer, reaching an array density of 625 spots/cm2. A 50-plexed immunoassay has been conducted to demonstrate the usability of the snap chip in multiplexed protein analysis. Limits of detection of 35 proteins are in the range of pg/mL.

We demonstrate a snap chip technology for performing cross-reactivity-free multiplexed sandwich immunoassays by simply snapping two slides. A snap apparatus is used for reliably transferring reagents from microarray-to-microarray. The snap chip can be used for any biochemical reactions requiring colocalization of different reagents without cross-contamination.

Çip için çapraz-reactivity ücretsiz ve gözcü-Alerjik çoğaltılmış sandviç uzun snap

Multiplexed protein analysis has shown superior diagnostic sensitivity and accuracy compared to single proteins. Antibody microarrays allow for thousands ...

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.

Gıda kaynaklı patojen tespit etmek için bir elektrokimyasal DNA biyoalgılayıcı gelişimi

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