この作品は、K INBRE P20GM103418、カンザス大豆委員会 (KSC/PSU 1663 年)、ACS PRF 56629 UNI7 と SS にすべて電源ユニット高分子化学スタートアップ資金によってサポートされます。ビデオと彼の顕著な仕事のため大学のビデオグラファー、ジェイコブ アンセルミ氏に感謝いたします。我々 もありがとう氏のロジャー ・ Heckert と夫人カタ Heckert 研究のための彼らの寛大なサポート。

1. 合成と多パラメトリック磁気蛍光ナノセンサー (MFnS) の機能化。
<ol><li>超常磁性酸化鉄の合成ナノ粒子 (IONPs) <ol><li>IONP 合成に備えて準備次の 3 ソリューション: ソリューション 1: した FeCl 3 (0.70 g) とした FeCl 2 H 2 O (2 mL)、ソリューション 2: NH 4 ああ (2.0 mL、13.4 M) H 2 O (15 mL)、ソリューション 3: ポリアクリル酸 (0.855 g) H 2 O (5 mL).</li>
		<li>は、ソリューション 1 に 2 M 塩酸 (HCl) の 90 μ l 添加しすぐに 875 rpm でボルテックスしながらソリューション 2 を混ぜます。解決策 3 を追加します。60 分のボルテックスを続ける</li>
		<li>1,620 x g に 20 分間遠心遠心 2,880 x g に 20 分間清</li>
		<li>は、透析による最終的な上澄みを浄化します。透析バッグに上澄みを追加 (MWCO 6 &#8722; 8 K)、リン酸緩衝生理食塩水 (PBS) が含まれているビーカーに配置 (pH = 7.4) とスピン バー。それはすべて 2-3 h. 新鮮なバッファーに置き換えて、12 時間浸してみましょう</li>
	</ol></li> <li>IONPs の表面にターゲット固有の抗体の共役 <ol><li>次の 4 つのソリューションを準備: ソリューション a: 5 mg 1-エチル - 3-(3-dimethylaminopropyl) 250 μ L MES のカルボジイミド塩酸塩 (EDC) [2-(N-morpholino) ethanesulfonic 酸] バッファー (0.1 M、pH = 6.8)、ソリューション b: 3 mg N-(NHS) の MES バッファーの 250 μ L (0.1 M、pH = 6.8)、IgG1、リン酸緩衝生理食塩水 (PBS) (pH 7.4) の 225 μ L で、大腸菌 mAb のソリューション c: 5 μ g、IONP ソリューション d: 4 ml (5.0 m モル) を 1 mL の PBS (pH 7.4)。</li>
		<li>追加 2 (N Morpholino) ethanesulfonic 酸 (MES) バッファー溶液をすぐに追加溶液ソリューション d 15 内の小さな単位で s、混合の各付加の後でソリューションを反転します</li>。
		<li>はすぐに 3 分間にわたって少しずつソリューション d ソリューション B を追加する開始します。各付加の後でソリューションを反転してミックスします</li>。
		<li>混合の各付加の後でソリューションを反転ソリューション C を 5 μ L の単位でソリューションに追加します</li>。
		<li>反応室温で 3 時間して一晩 4 ° C で孵化を続行するを許可します</li>。
		<li>PBS を使用する磁気の列を介して結果 Ab 共役 IONPs を浄化する (pH 7.4、決勝 [Fe] を = = 3.5 mmol) 任意の非抗体を削除します。
		<ol><li>PBS (1 mL) で洗浄磁気の列。磁気プレートに列を添付し、IONP ソリューションを追加します</li>。
			<li>は、PBS (1 mL) の磁気コラムを再度洗浄します。磁気プレートから磁気の列を削除します。PBS を列に追加し、ソリューションを収集します。4 ° C でストア</li></ol></li></ol></li> <li>カプセル化蛍光 1, 1 の &#39; - Dioctadecyl - 3,3,3 &#39; 3、&#39;-Tetramethylindocarbocyanine 過塩素酸塩 (DiI) 色素標識抗体 IONPs を使用してのポリアクリル酸 (PAA) のコーティングに、溶媒拡散法。
	<ol><li>、IONPs の 4 mL に追加滴 2.0 μ L で 1100 rpm 連続混合 DMSO の 100 μ L の DiI 色素 (2 mmol) の</li>。
		<li>[Fe] の最終的な鉄濃度を最終的な MFnS ソリューションの作成、PBS に対して得られた溶液 (12 h MWCO 6-8 KDa) を dialyze = 2 モル</li>。
	</ol></li> <li>MFnS 特性 <ol><li>吸光光度分析用プレート リーダー 595 で蛍光性の放出でカプセル化された DiI 染料を検出する nM</li>。
		<li>動的光散乱法による <ol><li>が平均サイズとは MFnS の表面電荷を決定する zetasizer に MFnS ソリューションのサンプルを配置します 。
			注: ここでは、平均サイズと機能 MFnS の表面電荷ことがわかった 77.09 nm と-22.3 mV、それぞれします</li>。
		</ol></li></ol></li></ol> 2。細菌の培養とストックに調製した溶液 <ol><li>細菌培養 <ol><li>水和物の凍結乾燥ペレット (E. 大腸菌 o157: h7) 栄養流体培養基の 1 mL にし、スープの追加 5 mL を追加します</li>。
		<li>寒天培地と連勝するには、このソリューションの適用 100 μ L を使用して滅菌接種ループ、37 ° C 24 時間で孵化が続く</li>
		<li>後 24 h、寒天板から隔離されたコロニーを選択し、15 mL の培養液を追加します。光学濃度値を監視しながら 4-6 時間、37 ° C で孵化させなさい (吸光度 600 nm).</li>
		<li>0.1 の光学密度値を取得した後、培養を停止し、シリアル希薄を実行します</li>。
	</ol></li> <li>シリアル希薄 <ol><li>8 つの滅菌遠心チューブに栄養流体培養基の追加 900 μ L.</li>
		<li>初の管 (希釈 10 -1) に細菌懸濁液の追加 100 μ L.</li>
		<li>最初から取る 100 μ L チューブ 10 -2 の希釈を取得する、2 番目に追加.残りのチューブ、最終希釈 10 -8 で終わるこのパターンを続ける</li>。
		<li>寒天培地プレートを分離し、37 で 24 時間インキュベート各チューブから転送 100 μ L ° C</li>
		<li>次の日は、各プレート上 (CFUs) の単位を形作っているコロニーをカウントします。〜 100 CFUs のプレートを選択します。さらに実験のための対応する希釈を使用 (100 μ L = 100 ~ CFUs). 
		注: ここにそれは 〜 100 CFUs の結果 10 -6 希釈します</li>。
	</ol></li></ol> 3。迅速な検出の E. 大腸菌 o157: h7 MFnS を使用して <ol><li>スパイク 1 ~ 100 CFU 範囲の結果 10 -6 細菌の在庫の増加額で様々 な PBS ソリューション (1 X、pH 7.4 では、300 μ L)。MFnS の一貫した量を追加 (100 μ L) これらのソリューションにします</li>。
	<li>PBS (1 X、pH 7.4 では、300 μ L) のみが含まれている 1 つのベースライン ソリューションを作成し、MFnS (100 μ L).</li>
	<li>37 ° C で 30 分のためのソリューションをインキュベートし、部屋の温度を冷ますします</li>。
	<li>磁気 relaxometer (0.47 テスラ) と各ソリューションの CFUs 基準緩和時間 (T2) の変更を記録する個々 のソリューションを転送します。
	<ol><li>T2 値の変化を記録、PBS と MFnS のみを含むベースライン ソリューションの T2 値を測定することによって開始します。
		<ol><li>磁気でソリューションの場所 relaxometer。それぞれのソフトウェアを開き、選択、&#34; T2 緩和 &#34; 設定を押し &#34; 測定 &#34;</li></ol></li> <li>様々 なスパイクが追加のソリューションの T2 値を測定基準 T2、のコレクションを次。細菌の濃度. 
		注意: T2 に変更、ベースライン スパイク T2 から減算 T2 に相当します</li>。
	</ol></li> <li>磁気 relaxometer からのサンプルを削除し 2880 x g で 10 分間のチューブを遠心分離</li>
	<li>上清を捨て、100 μ L の PBS (1 X、pH 7.4) の細菌のペレットを再懸濁します</li>。
	<li>96 ウェル プレートと 595 でのレコードの蛍光強度をそれぞれの巻き上がりを追加 80 μ L nM 。
	注: テスト繰り返すことができる結果のセクションで説明するように、湖の水、ミルクなど、異なる溶媒を用いた</li></ol>

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</ol>

このナノテクノロジーの実証済みのアプリケーションは、まだ FDA によって承認されていません。

このプロトコルは、単に完全に機能の MFnS を生成する設計されている可能な限り。ただし、プロトコルの変更、ユーザーの目的に応じて、役に立つかもしれない多くの重要なポイントがあります。たとえば、異なった抗体の使用は他の多くの病原体の対象になります。さらに、このプロトコルは抗体分子をターゲットとしての使用に限定ではありません。ホスト細胞?...

両方の細菌汚染度の永続的な発生市販食品と水の源はますます急速なと特定の診断プラットフォームの必要性を作成しました。1,2より一般的な細菌汚染食料と水の汚染のための責任の一部はサルモネラ、黄色ブドウ球菌、リステリア菌、腸炎ビブリオ、赤痢菌、細菌、大腸菌属です。3,4多くの場合これらの病原体が細菌汚染の結果熱、コレラ、胃腸炎、下痢などの症状。4しばしば水源の汚染が十分にろ過された水へのアクセスなしでコミュニティに抜本的な不利な影響と食品汚染が病気や製品のリコールの努力の偉大な数につながっています。5,6
細菌汚染に起因する病気の発生を減らすために、水と食品を効率的にスキャン販売または消費する前に方法を開発する努力の数がずっとあります。1,7,8,9,10 12ループ lamp 法 (11,, elisa 法、PCR などの3手法ランプ)13,14 15、16,17,18,19,20,21、 22,23,24は、最近様々 な病原体の検出に使用されています。伝統的な細菌の培養方法と比較して、これらの技術、特異性と時間に関してはるかに多く効率的です。しかし、これらの技術はまだ偽陽性とネガ、複雑な手順、コストと戦います。1,3,25細菌検出のための代替方法として多重磁気蛍光ナノセンサー (MFnS) を提案する、まさにこの理由のためです。
これらナノセンサーでの磁気緩和と迅速かつ適確なデュアル検出プラットフォームを可能にする蛍光のモダリティのペアリング方法一緒に一意に。E. 大腸菌o157: h7 を使用すると、サンプルの汚染物質として、分以内として少し 1 CFU を検出する MFnS の能力を発揮します。病原体特異抗体は特異性を上げるため、磁気や蛍光灯のモダリティの組み合わせにより、検出の両方の低と高の汚染範囲の細菌汚染の定量化。16細菌汚染の場合、ナノセンサー、細菌病原体特異的抗体のターゲットの能力のため周辺群発地震します。磁気ナノセンサーと細菌の間のバインドは、磁気鉄のコアと周辺の水のプロトン間の相互作用を制限します。これは磁気 relaxometer で記録された T2 緩和時間の増加を引き起こします。ソリューションにおける細菌の濃度が上昇すると、T2 値が小さい結果、細菌数の増加など、ナノセンサーを分散します。逆に、病原体を直接バインド ナノセンサーの数が増えたため、細菌の濃度に比例した蛍光性の放出が増えます。サンプルの遠心分離と分離細菌のペレットのみ任意の浮遊ナノセンサーを取り外して直接関連付けの数と蛍光性の放出、細菌に直接接続されているナノ粒子を節約します。ソリューションに存在する細菌。このメカニズムの概略は、図 1で表されます。
この MFnS プラットフォームは、ポイント ・ オブ ・ ケア スクリーニング低コスト ・ ポータブルの特性で、心で設計されています。MFnS は常温で安定しているし、細菌汚染の正確な検出のための非常に低濃度で必要なだけです。さらに、合成後に、、MFnS の使用は簡単、分野で訓練を受けた専門家の使用を必要としません。最後に、この診断プラットフォームにより高度なカスタマイズをターゲットに、さまざまな設定のすべての種類の病原体を検出する 1 つのプラットフォームを使用可能性があります、この手段を提供します。

<table><tbody><tr><td>Ferrous Chloride Tetrahydrate</td><td>Fisher Scientific</td><td>I90-500</td><td></td></tr><tr><td>Ferric Chloride Hexahydrate</td><td>Fisher Scientific</td><td>I88-500</td><td></td></tr><tr><td>Ammonium Hydroxide</td><td>Fisher Scientific</td><td>A669S-500</td><td></td></tr><tr><td>Hydrochloric Acid</td><td>Fisher Scientific</td><td>A144S-500</td><td></td></tr><tr><td>Polyacryllic Acid</td><td>Sigma-Aldrich</td><td>323667-100G</td><td></td></tr><tr><td>EDC</td><td>Thermofisher Scientific</td><td>22980</td><td></td></tr><tr><td>NHS</td><td>Fisher Scientific</td><td>AC157270250</td><td></td></tr><tr><td>Anti-E. coli O111 antibody&amp;nbsp;</td><td>sera care</td><td>5310-0352</td><td></td></tr><tr><td>Anti-E. coli O157:H7 antibody [P3C6]&amp;nbsp;</td><td>Abcam</td><td>ab75244</td><td></td></tr><tr><td>DiI Stain</td><td>Fisher Scientific</td><td>D282</td><td></td></tr><tr><td>Nutrient Broth</td><td>Difco</td><td>233000</td><td></td></tr><tr><td>Freeze-dried E. coli O157:H7 pellet</td><td>ATCC</td><td>700728</td><td></td></tr><tr><td>Magnetic Relaxomteter&amp;nbsp;</td><td>Bruker</td><td>mq20</td><td></td></tr><tr><td>Zetasizer</td><td>Malvern</td><td>NANO-ZS90</td><td></td></tr><tr><td>Plate Reader&amp;nbsp;</td><td>Tecan</td><td>Infinite M200 PRO</td><td></td></tr><tr><td>Magnetic Column&amp;nbsp;</td><td>QuadroMACS</td><td>130-090-976</td><td></td></tr><tr><td>Centrifuge</td><td>Eppendorf</td><td>5804 Series</td><td></td></tr><tr><td>Centrifuge (accuSpin Micro 17)</td><td>Fisher Scientific</td><td>13-100-676</td><td></td></tr><tr><td>Floor Model Shaking Incubator</td><td>SHEL LAB</td><td>SSI5</td><td></td></tr><tr><td>Analytical Balance</td><td>Metler Toledo</td><td>ME104E</td><td></td></tr><tr><td>Digital Vortex Mixer</td><td>Fisher Scientific</td><td>02-215-370</td><td></td></tr><tr><td>Open-Air Rocking Shaker</td><td>Fisher Scientific</td><td>02-217-765</td><td></td></tr></tbody></table>

foodborne pathogen screening using magneto-fluorescent nanosensor: rapid detection of e. coli o157:h7

腸管出血性大腸菌o157: h7 は、水性の両方にリンクされている媒介する病気と食料と水上映方法にも関わらず脅威は現在使用されるまま。従来の細菌検出方法、ポリメラーゼ連鎖反応 (PCR)、酵素抗体法 (ELISA) 特に病原性の汚染物質を検出できるよう彼らが豊富なサンプル準備と待機時間が長く必要です。さらに、これらのプラクティスは洗練された実験室の器械と設定、要求し、訓練を受けた専門家によって実行される必要があります。ここで、ナノ粒子ベースのプラットフォームの磁気および蛍光パラメーターの一意の組み合わせを備えて簡単診断技術のためのプロトコルを提案します。提案多重磁気蛍光ナノセンサー (MFnS) は、大腸菌o157: h7 汚染としてはほとんど 1 コロニー形成単位未満 1 時間以内のソリューションに存在を検出できます。牛乳など複雑なメディアで高機能を維持する MFnS の能力をさらに、湖の水を確認します。その他特異性アッセイも同じような細菌種の存在下でも、特定のターゲット細菌のみを検出する MFnS の能力を発揮する使用されました。検出の両方の初期と後期段階の汚染検出でその高性能を出展、濃度の広い範囲の病原体汚染の定量化により、磁気や蛍光灯のモダリティのペアリングします。有効性、手頃な価格、および、MFnS の移植性から作ることがポイント ・ オブ ・ ケア スクリーニング細菌汚染のための理想的な候補の設定の広い範囲で市販加工食品に水生の貯水池。

MFnS の作用機序は、図 1で表されます。細菌汚染物質の表面周囲の MFnS のクラスタ リングは、MFnS の magnetic cores と周囲の水素原子核の相互作用を妨げます。このクラスタ リング、磁気緩和の結果として値が増加します。細菌汚染の濃度が増えると、クラスタ リングが縮小し、T2 値の変動が減少します。したがって、蛍光のモダリティの追加が重要です。細菌の濃度が増...

Watch this Scientific Journal Video about Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7 at JoVE.com

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

このプロトコルの全体的な目標はポータブル、コスト効果の高い、機能的なナノセンサーを合成する、特にターゲットに磁気緩和と蛍光発光モダリティの組み合わせにより病原性細菌の迅速検出。

E. 大腸菌o157: h7 の食品由来病原体のスクリーニングを使用して磁気蛍光ナノセンサー: 迅速検出

Enterohemorrhagic Escherichia coli O157:H7 has been linked to both waterborne and foodborne illnesses, and remains a threat despite the food- and ...

foodborne-pathogen-screening-using-magneto-fluorescent-nanosensor

Research

JoVE Journal

Immunology and Infection

Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

7.6K ビュー.  Pittsburg State University. このプロトコルの全体的な目標はポータブル、コスト効果の高い、機能的なナノセンサーを合成する、特にターゲットに磁気緩和と蛍光発光モダリティの組み合わせにより病原性細菌の迅速検出。

ビデオ: Foodborne Pathogen Screening Using Magneto-fluorescent Nanosensor: Rapid Detection of E. Coli O157:H7

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

蛍光性DNAzymesを使用した細菌の検出

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

免疫・感染学

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

を光学的に検出<em&gt; E.大腸菌の</emメソポーラスケイ素バイオセンサーによる&gt;バクテリア

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

生物工学

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described.
Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 (MNP@SiO2) using the St&#246;ber process followed by functionalization with APTES (MNP@SiO2@NH2). Then, feroxamine was conjugated with the MNP@SiO2@NH2 by carbodiimide coupling to give MNP@SiO2@NH2@Fa. The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.

This work describes protocols for the preparation of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine using a succinyl moiety as a linker. A deep structural characterization description and a capture bacteria assay using Y. enterocolitica for all the intermediate nanoparticles and the final conjugate are also described in detail.

機能化された磁気ナノ粒子の合成、その結合とゼリロフォア・フェロキサミンの評価

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with ...

化学

Rapid Homogeneous Detection of Biological Assays Using Magnetic Modulation Biosensing System

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or separation step. When the IL-8 target was present, a 'sandwich'-based assay attached magnetic beads with IL-8 capture antibody to streptavidin coupled fluorescent protein via the IL-8 target and a biotinylated IL-8 antibody. The magnetic beads are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The fluorescent proteins, which are attached to the magnetic beads are condensed into the detection area and their movement in and out of an orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. The magnetic modulation biosensing system was previously used to detect the coding sequences of the non-structural Ibaraki virus protein 3 (NS3) complementary DNA (cDNA) [2]. The techniques that are demonstrated in this work for external manipulation and condensation of particles may be used for other applications, e.g. delivery of magnetically-coupled drugs in-vivo or enhancing the contrast for in-vivo imaging applications.

Magnetic modulation biosensing system is utilized to rapidly, sensitively and simply detect biological assays, such as DNA molecules and proteins.

磁気変調バイオセンシングシステムを用いた生物学的アッセイの迅速な均質な検出

A magnetic modulation biosensing system (MMB) [1,2] rapidly and homogeneously detected biological targets at low concentrations without any washing or ...

生物学

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.

食品由来病原体を検出する電気化学的 DNA のバイオ センサーの開発

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

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and allostasis. Moreover, dysregulated protease activity drives pathogenesis and is a functional biomarker of diseases such as cancer; therefore, the ability to detect protease activity in vivo may provide clinically relevant information for biomedical diagnostics. The goal of this protocol is to create nanosensors that probe for protease activity in vivo by producing a quantifiable signal in urine. These protease nanosensors consist of two components: a nanoparticle and substrate. The nanoparticle functions to increase circulation half-life and substrate delivery to target disease sites. The substrate is a short peptide sequence (6-8 AA), which is designed to be specific to a target protease or group of proteases. The substrate is conjugated to the surface of the nanoparticle and is terminated by a reporter, such as a fluorescent marker, for detection. As dysregulated proteases cleave the peptide substrate, the reporter is filtered into urine for quantification as a biomarker of protease activity. Herein we describe construction of a nanosensor for matrix metalloproteinase 9 (MMP9), which is associated with tumor progression and metastasis, for detection of colorectal cancer in a mouse model.

Nanosensors to Detect Protease Activity In Vivo for Noninvasive Diagnostics

Proteases are tightly regulated enzymes involved in fundamental biological processes, and dysregulated protease activity drives progression of complex diseases such as cancer. This method&#39;s goal is to create nanosensors that measure protease activity in vivo by producing a cleavage signal that is detectable from host urine and discriminates disease.

非侵襲的診断のプロテアーゼ活動In Vivo検出するナノセンサー

Proteases are multi-functional enzymes that specialize in the hydrolysis of peptide-bonds and control broad biological processes including homeostasis and ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

サルモネラ属菌のスクリーニングのための高スループット プラットフォーム/赤痢菌属

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

Polymerase chain reaction (PCR) is a traditional method employed for the amplification of a target gene that has played an important role in biomolecular diagnostics. However, traditional PCR is very time-consuming because of the low-temperature variation efficiency. This work proposes a continuous-flow-PCR (CF-PCR) system based on a microfluidic chip. The amplification time can be greatly reduced by running the PCR solution into a microchannel placed on heaters set at different temperatures. Moreover, as capillary electrophoresis (CE) is an ideal way to differentiate positive and false-positive PCR products, a CE system was built to achieve efficient separation of the DNA fragments. This paper describes the process of amplification of Escherichia coli (E. coli) by the CF-PCR system built in-house and the detection of the PCR products by CE. The results demonstrate that the target gene of E. coli was successfully amplified within 10 min, indicating that these two systems can be used for the rapid amplification and detection of nucleic acids.

Amplification of Escherichia coli in a Continuous-Flow-PCR Microfluidic Chip and Its Detection with a Capillary Electrophoresis System

This protocol describes how to build a continuous-flow-polymerase chain system based on a microfluidic chip and how to build a capillary electrophoresis system in the lab. It presents a simple method for the analysis of nucleic acids in the lab.

連続フローPCRマイクロ流体チップにおける大 腸菌 の増幅とキャピラリー電気泳動システムによる検出

Polymerase chain reaction (PCR) is a traditional method employed for the amplification of a target gene that has played an important role in biomolecular ...

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. coli O157:H7 by targeting ORF Z3276. With this assay, we can detect as low as a few copies of the genome of DNA of E. coli O157:H7. The sensitivity and specificity of the assay were confirmed by intensive validation tests with a large number of E. coli O157:H7 strains (n = 369) and non-O157 strains (n = 112). Furthermore, we have combined propidium monoazide (PMA) procedure with the newly developed qPCR protocol for selective detection of live cells from dead cells. Amplification of DNA from PMA-treated dead cells was almost completely inhibited in contrast to virtually unaffected amplification of DNA from PMA-treated live cells. Additionally, the protocol has been modified and adapted to a 96-well plate format for an easy and consistent handling of a large number of samples. This method is expected to have an impact on accurate microbiological and epidemiological monitoring of food safety and environmental source.

Detection of Live Escherichia coli O157:H7 Cells by PMA-qPCR

A qPCR assay was developed for detection of Escherichia coli O157:H7 targeting a unique genetic marker, Z3276. The qPCR was combined with propidium monoazide (PMA) treatment for live cell detection. This protocol has been modified and adapted to a 96-well plate format for easy and consistent handling of numerous samples

ライブの検出<em&gt;エシェリヒア·コリ</em&gt; O157：PMA-qPCRによりH7細胞

A unique open reading frame (ORF) Z3276 was identified as a specific genetic marker for E. coli O157:H7. A qPCR assay was developed for detection of E. ...

Colorimetric Detection of Bacteria Using Litmus Test

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. Such methods need to be user-friendly and produce reliable results that can be easily interpreted by both specialists and non-professionals. The litmus test for pH is simple, quick, and effective as it reports the pH of a test sample via a simple color change. We have developed an approach to take advantage of the litmus test for bacterial detection. The method exploits a bacterium-specific RNA-cleaving DNAzyme to achieve two functions: recognizing a bacterium of interest and providing a mechanism to control the activity of urease. Through the use of magnetic beads immobilized with a DNAzyme-urease conjugate, the presence of bacteria in a test sample is relayed to the release of urease from beads to solution. The released urease is transferred to a test solution to hydrolyze urea into ammonia, resulting in an increase of pH that can be visualized using the classic litmus test.

We describe a protocol for colorimetric detection of E. coli using a modified litmus test that takes advantage of an RNA-cleaving DNAzyme, urease, and magnetic beads.

リトマス試験を用いて細菌の比色検出

There are increasing demands for simple but still effective methods that can be used to detect specific pathogens for point-of-care or field applications. ...

E. 大腸菌o157: h7 の食品由来病原体のスクリーニングを使用して磁気蛍光ナノセンサー: 迅速検出

要約

さらに動画を探す

蛍光性DNAzymesを使用した細菌の検出

を光学的に検出

機能化された磁気ナノ粒子の合成、その結合とゼリロフォア・フェロキサミンの評価

磁気変調バイオセンシングシステムを用いた生物学的アッセイの迅速な均質な検出

食品由来病原体を検出する電気化学的 DNA のバイオセンサーの開発

非侵襲的診断のプロテアーゼ活動In Vivo検出するナノセンサー

サルモネラ属菌のスクリーニングのための高スループットプラットフォーム/赤痢菌属

連続フローPCRマイクロ流体チップにおける大腸菌の増幅とキャピラリー電気泳動システムによる検出

ライブの検出

リトマス試験を用いて細菌の比色検出

E. 大腸菌o157: h7 の食品由来病原体のスクリーニングを使用して磁気蛍光ナノセンサー: 迅速検出

要約

さらに動画を探す

蛍光性DNAzymesを使用した細菌の検出

を光学的に検出

機能化された磁気ナノ粒子の合成、その結合とゼリロフォア・フェロキサミンの評価

磁気変調バイオセンシングシステムを用いた生物学的アッセイの迅速な均質な検出

食品由来病原体を検出する電気化学的 DNA のバイオ センサーの開発

非侵襲的診断のプロテアーゼ活動In Vivo検出するナノセンサー

サルモネラ属菌のスクリーニングのための高スループット プラットフォーム/赤痢菌属

連続フローPCRマイクロ流体チップにおける大 腸菌 の増幅とキャピラリー電気泳動システムによる検出

ライブの検出

リトマス試験を用いて細菌の比色検出

食品由来病原体を検出する電気化学的 DNA のバイオセンサーの開発

サルモネラ属菌のスクリーニングのための高スループットプラットフォーム/赤痢菌属

連続フローPCRマイクロ流体チップにおける大腸菌の増幅とキャピラリー電気泳動システムによる検出