Name: a rapid and chemical-free hemoglobin assay with photothermal angular light scattering
Uploaded: 2016-12-07T15:00:00.000+00:00
Duration: 5 min 18 s
Description: Watch this Scientific Journal Video about A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering at JoVE.com

This research was supported by the research programs of the National Research Foundation of Korea (NRF) (NRF-2015R1A1A1A05001548 and NRF-2015R1A5A1037668).

血液サンプルを用いた実験では、関連する法律や制度のガイドラインに準拠して行きました。サンプルを取得し、教育機関での臨床試験で処理されていた残留血液サンプルでした。 1. PT-AS光のセットアップ注：一つは初期PT-ASのセットアップのための空のマイクロキャピラリーチューブを使用することができます。 <ol><li>それぞれ、200および330ミクロンの内径と外径と空のマイクロキャピラリーチューブをマウントし、毛細管器具に大きな5〜cm以上の長さ。市販のファイバ固定具は、チューブ固定具として使用することができます。 </li><li>しっかりと650 nmのレーザーポインターを固定、 すなわち 、プローブ光源、毛細管を照明します。プローブビームは、毛細管よりも大きくする必要があります。角度の周期パターンを観察するために毛細管の背後にある画面（ 例えば 、ホワイトペーパー）を配置します。 </li><li>検出部は、直接scattをキャプチャするウェブカメラのいずれかのレンズをはずしてパターンをering。プローブビーム方向に対して25から35°の角度で毛細管の背後にあるウェブカメラを置きます。毛細管によって生成される角度の周期パターンが検出器（ 図1）を用いて測定することができることを確認してください。イメージセンサが適切に配置されたときに、イメージセンサの途中で角度周期パターンを観察します。 </li><li>毛細管を照明する位置532nmのPTの励起光源。限りPT励起光が毛細管のプローブビームと重複し、直接検出器に到達しないように任意の角度で位置PT光源を、。それはRIより大きな変化をもたらすように、通常、高い光パワーを用いて、血液サンプルのPTの励起は、PT-AS感度を向上させます。 <ol><li>採用PT励起光源の最も高い光パワーを使用してください。また、PTの励起光はキャピラリチューブにプローブ光と重なることを確認してください。 PT励起光のビームサイズを使用少なくとも2倍、そのプローブ光の全プローブ体積を加熱します。 </li></ol></li><li> 532 nmの光を遮断し、唯一650 nmのプローブ光を測定する検出器の前にロングパスフィルタを置きます。 </li><li>毛細管を照射する前に、PTの励起光の経路内に光チョッパをインストールします。光チョッパは、PTの励起光強度を変調するために使用されます。 </li></ol> 2.血液サンプルの調製<ol><li>エチレンジアミン四酢酸採血管に貧血状態で、新鮮な全血の6ミリリットルを描き、よくサンプルを混ぜます。他の処理は必要ありません。 </li><li>凝固を防ぐために、抽出の24時間以内にPT-ASセンサを用いて血液試料を測定します。 </li></ol> 3. PT-AS測定プロトコル<ol><li>測定するために血液サンプルを用いて、マイクロキャピラリーチューブをロードします。血液の中にチューブを配置することにより、毛細管現象を介して血液と毛細管を埋めます十分な。測定に必要な最小試料容量は、毛細管の内径及びプローブビームサイズによって決定されます。 <ol><li> 200μmの内径を有する管を採用しています。プローブビームのサイズは、測定が&gt; 63ナノリットルの試料容量を用いて行うことができることを示唆している、代表的な結果は2mmでした。 </li></ol></li><li>フィクスチャ内の指定位置に毛細管をマウントします。 </li><li>血液ロードされたマイクロキャピラリーチューブを照明するために650 nmのプローブレーザをオンにします。角度の周期パターンは、ウェブカメラで観察する必要があります。 </li><li>チューブを照らすために532-nmのPTの励起レーザーをオンにします。 </li><li> 2 HzでPTの励起光の強度を変調する光チョッパを実行します。 注：この動作条件の選択のための理論的根拠は、ディスカッションおよびKim らに記載されています。 12。 <ol><li>光チョッパのモーターヘッドアセンブリにチョッパーホイールを装着システム。 </li><li>チョッパ制御ボックスをオンにして、変調周波数を設定するために、コンソールにコントロールノブを使用します。 </li><li>コントロールノブを使用してチョッパーを実行します。 </li></ol></li><li> MPEG-4（MP4）形式で5秒間のウェブカメラを経由して変動する散乱パターンを記録します。 </li></ol> 4.信号処理注：PT-AS信号処理は、実験室開発MATLABコードを使用して行きました。 <ol><li>画像を抽出するために、ビデオファイルをロードします。各画像について、垂直方向[ 図2（B、C）]に沿った画素値の平均を計算することによって平均散乱パターンを得た[ 図2（a）は代表的な画像を参照]。 </li><li>フーリエ変換を平均散乱パターンの変換を評価し、ピーク空間周波数で位相を計算します。すべての記録画像のすべてのフレームのためにこれらの操作を実行します。 </li><li>すべての画像から得られた位相値を用いて、時間的な位相をプロット変動〔 図2（D）]。位相がPTの変調周波数で変動することに注意してください。時間領域における位相変動の変換、および変調周波数での大きさを得るフーリエ変換を取ります。この信号は、PT-AS信号〔 図2（E）]と呼ばれます。 </li><li>プロトコル5で得られた検量線を用いて、[Hbの】対応にそのPT-AS信号を変換して、血液サンプルの[ヘモグロビン]を測定します。 </li></ol> 5. PT-ASキャリブレーション<ol><li> （ - 18グラム/デシリットル例えば 、0）を均一PT-ASセンサの検出範囲に分布している[ヘモグロビン]の値を持つ、血液サンプルを準備します。 </li><li>キャリブレーションの前に、参照血液分析器を使用して、サンプルの[Hbの]の値を定量化します。サンプルのPT-AS信号を測定します。 </li><li>線形最小二乗を行うことにより、PT-AS信号に[Hbの]に関する校正曲線を導出エクスペリの、B + [ヘモグロビン] = A [PT-AS信号]、フィットLが得られます。動作条件は[ヘモグロビン]との関係は 、 表1に指定され、PT-AS信号は、[ヘモグロビン] = 5.13 [PT-AS信号]であることがわかった- 0.09。線形フィットを実行するためにMATLABコードを使用してください。 </li></ol>

<ol>
	<li>Mokken, F. C., Kedaria, M., Henny, C. P., Hardeman, M., Gelb, 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+clinical+importance+of+erythrocyte+deformability,+a+hemorrheological+parameter.">The clinical importance of erythrocyte deformability, a hemorrheological parameter.</a> Ann. Hematol. 64 (3), 113-122 (1992).</li><li>Rosenblit, 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=Evaluation+of+three+methods+for+hemoglobin+measurement+in+a+blood+donor+setting.">Evaluation of three methods for hemoglobin measurement in a blood donor setting.</a> Sao Paulo Medical Journal. 117 (3), 108-112 (1999).</li><li>Van Kampen, E., Zijlstra, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standardization+of+hemoglobinometry+II.+The+hemiglobincyanide+method.">Standardization of hemoglobinometry II. The hemiglobincyanide method.</a> Clin. Chim. Acta. 6 (4), 538-544 (1961).</li><li>Billett, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemoglobin+and+hematocrit.">Hemoglobin and hematocrit.</a> Clinical Methods: The History, Physical, and Laboratory Examinations. 3, (1990).</li><li>Kuenstner, J. T., Norris, K. H., McCarthy, W. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+hemoglobin+in+unlysed+blood+by+near-infrared+spectroscopy.">Measurement of hemoglobin in unlysed blood by near-infrared spectroscopy.</a> Appl. Spectrosc. 48 (4), 484-488 (1994).</li><li>Zwart, A., 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=A+multi-wavelength+spectrophotometric+method+for+the+simultaneous+determination+of+five+haemoglobin+derivatives.">A multi-wavelength spectrophotometric method for the simultaneous determination of five haemoglobin derivatives.</a> Clin. Chem. Lab. Med. 19 (7), 457-464 (1981).</li><li>Kwak, B. S., 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=Direct+measurement+of+the+in+vitro+hemoglobin+content+of+erythrocytes+using+the+photo-thermal+effect+of+the+heme+group.">Direct measurement of the in vitro hemoglobin content of erythrocytes using the photo-thermal effect of the heme group.</a> Analyst. 135 (9), 2365-2371 (2010).</li><li>Lapotko, D., Lukianova, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+micro-bubbles+in+cells.">Laser-induced micro-bubbles in cells.</a> International Journal of Heat Mass Transfer. 48 (1), 227-234 (2005).</li><li>Lapotko, D. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser-induced+bubbles+in+living+cells.">Laser-induced bubbles in living cells.</a> Lasers in surgery and medicine. 38 (3), 240-248 (2006).</li><li>Lapotko, D. O., Romanovskaya, T. y. R., Shnip, A., Zharov, V. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photothermal+time-resolved+imaging+of+living+cells.">Photothermal time-resolved imaging of living cells.</a> Lasers in surgery and medicine. 31 (1), 53-63 (2002).</li><li>Yim, 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=Photothermal+spectral-domain+optical+coherence+reflectometry+for+direct+measurement+of+hemoglobin+concentration+of+erythrocytes.">Photothermal spectral-domain optical coherence reflectometry for direct measurement of hemoglobin concentration of erythrocytes.</a> Biosens. Bioelectron. 57, 59-64 (2014).</li><li>Kim, U., 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=Capillary-scale+direct+measurement+of+hemoglobin+concentration+of+erythrocytes+using+photothermal+angular+light+scattering.">Capillary-scale direct measurement of hemoglobin concentration of erythrocytes using photothermal angular light scattering.</a> Biosens. Bioelectron. 74, 469-475 (2015).</li><li>S&#0248;rensen, H. S., Larsen, N. B., Latham, J. C., Bornhop, D. J., Andersen, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+biosensing+based+on+interference+from+light+scattering+in+capillary+tubes.">Highly sensitive biosensing based on interference from light scattering in capillary tubes.</a> Appl. Phys. Lett. 89 (15), 151108 (2006).</li><li>Swinney, K., Markov, D., Bornhop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasmall+volume+refractive+index+detection+using+microinterferometry.">Ultrasmall volume refractive index detection using microinterferometry.</a> Rev. Sci. Instrum. 71 (7), 2684-2692 (2000).</li><li>Tarigan, H. J., Neill, P., Kenmore, C. K., Bornhop, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capillary-scale+refractive+index+detection+by+interferometric+backscatter.">Capillary-scale refractive index detection by interferometric backscatter.</a> Anal. Chem. 68 (10), 1762-1770 (1996).</li><li>Bornhop, D. 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=Free-solution,+label-free+molecular+interactions+studied+by+back-scattering+interferometry.">Free-solution, label-free molecular interactions studied by back-scattering interferometry.</a> science. 317 (5845), 1732-1736 (2007).</li><li>Yang, X., 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=Simple+paper-based+test+for+measuring+blood+hemoglobin+concentration+in+resource-limited+settings.">Simple paper-based test for measuring blood hemoglobin concentration in resource-limited settings.</a> Clin. Chem. 59 (10), 1506-1513 (2013).</li><li>Zhu, H., 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=Cost-effective+and+rapid+blood+analysis+on+a+cell-phone.">Cost-effective and rapid blood analysis on a cell-phone.</a> Lab Chip. 13 (7), 1282-1288 (2013).</li><li>Poga&#269;nik, L., Franko, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+organophosphate+and+carbamate+pesticides+in+vegetable+samples+by+a+photothermal+biosensor.">Detection of organophosphate and carbamate pesticides in vegetable samples by a photothermal biosensor.</a> Biosens. Bioelectron. 18 (1), 1-9 (2003).</li></ol>

No conflict of interest is declared.

PT-ASセンサは、未処理の血液サンプルの直接【のHb]測定可能な全光学的方法を表します。この方法は、赤血球中のヘモグロビン分子の固有のPT応答を使用して、血液中の[Hbを]定量化します。 532 nmの光による照明下で、ヘモグロビン分子が光エネルギーを吸収して熱を発生します。得られた温度上昇は、血液試料のRIを変更します。 BSIの高RI感度は、血液中のこのRIの変化を測定するために利?...

血液検査は、一般的に全体的な人間の健康を評価し、特定の疾患に関連するバイオマーカーを検出するために行われます。例えば、血液中のコレステロール濃度は、密接に、心血管疾患および膵炎に関連する高脂血症のための基準として機能します。グルコースレベルは、糖尿病性ケトアシドーシス及び高血糖高浸透圧症候群などの合併症に関連付けられているように、血糖の内容は、頻繁に測定すべきです。例えばマラリア、ヒト免疫不全ウイルスおよび後天性免疫不全症候群などの深刻な病気は、血液検査によって診断され、赤血球、血小板、および白血球を含む血液成分の定量化は、膵臓及び腎臓疾患のスクリーニングを可能にしています。 ヘモグロビン（Hb）は、血液の重要な成分は、赤血球の約96％を占め、そして人間の臓器に酸素を輸送します。その質量濃度（[ヘモグロビン]）の有意な変化は、私を指示することができますtabolic変化、肝胆道疾患、および神経、心血管および内分泌疾患1。 【ヘモグロビン従って日常の血液検査で測定されます。特に、貧血患者、透析患者、および妊娠中の女性は強く重要なタスク2として[ヘモグロビン]を監視することをお勧めします。 様々な[ヘモグロビン]の検出方法は、このように開発されています。ヘモグロビンシアン化方法、[ヘモグロビン]定量化のための最も一般的な技術の一つは、赤血球3の脂質二重層を破壊するためにシアン化カリウム（KCN）を用います。 540 nmの周りの化学展示高い吸収によって生成されるシアン化ヘモグロビン;従って、[ヘモグロビン]測定は、比色分析を介して行うことができます。この方法は、広くそのシンプルさのため採用したが、採用化学物質（ 例えば 、KCNおよびジメチルラウリルアミンオキシド）は、ヒトや環境への毒性がありますさ。ヘマトクリットスキームは、全血の体積に比べて赤血球の体積比を測定します梅の遠心分離を介して、しかし、それは比較的大きな血液量（50-100μl）を4が必要です。分光測光法は、[ヘモグロビン]正確に任意の化学物質なしで測定したが、複数の波長と大きな血液量で測定が5,6を必要としています。同様に、[ヘモグロビン]測定するためのいくつかの光学的方法は、光散乱に基づく検出方法を含む提案されてきたが、その測定精度は、理論的な血液モデルの精度に強く依存します。 光熱（PT）に基づいて、これらの制限を克服するために、[ヘモグロビン]検出方法Hbの効果は、最近7に提案されています。主に酸化鉄から構成さHbは、532 nmの光を吸収し熱8-10に光エネルギーに変換します。このPTの温度上昇は、血液サンプルの屈折率（RI）の変化を測定することによって光学的に検出することができます。イムら。就業スペクトルドメイン光コヒーレンスreflectometryは血液収容室11内のPT光路長の変化を測定します。この方法は、無農薬やダイレクト[Hbの]測定を可能にしているが、分光計および干渉計構成の使用は、その小型化の妨げとなる場合があります。我々は最近、代替[ヘモグロビン]検出方法を提示し、デバイスの微細化12に適している光熱角度光散乱（PT-AS）センサーを、と呼ばれます。 PT-ASセンサは、毛細管内部の血液試料のRIにおけるPTの変化を測定するために後方散乱干渉（BSI）の高RI感度を利用します。 BSIは、様々なソリューション13-15のRIを測定し、自由溶液16中の生化学的相互作用をモニターするために利用されてきました。 PT-ASセンサはBSIと同様の光学配置を採用したが、血液試料中のRIのPTの上昇を測定する光熱励起設定を組み合わせます。 BSIおよびPT-ASセンサの動作原理は、他の場所で詳細に記載されています<sup&gt; 12,15。 PT-ASセンサは、広い検出範囲（0.35から17.9までグラム/デシリットル）で高感度[ヘモグロビン]測定値を示し、&lt;100nlのサンプル容量で動作することができます。血液サンプルのないプレコンディショニングを必要とせず、測定時間は、約5秒です。ここでは、実験と詳細な測定手順について説明します。代表的なPT-ASの結果は、貧血患者から血液サンプルを使用して提供され、その結果は、PT-ASセンサの精度を評価するための血液分析器からのものと比較されます。

<table><tbody><tr><td>650 nm laser pointer</td><td>LASMAC</td><td>LED-1</td><td>Probe light</td></tr><tr><td>Hollow round glass capillaries</td><td>VitroCom</td><td>CV2033</td><td>Blood sample container</td></tr><tr><td>Webcam</td><td>Logitech</td><td>C525</td><td>CMOS optical sensor</td></tr><tr><td>Optical chopper system</td><td>Thorlabs</td><td>MC2000-EC</td><td>Optical chopper</td></tr><tr><td>Plastic long-pass filter</td><td>Edmund Optics</td><td>#43-942</td><td>To reject 532-nm PT excitation light</td></tr><tr><td>Fiber clamp</td><td>Thorlabs</td><td>SM1F1-250</td><td>Capillary tube fixture</td></tr><tr><td>EDTA coated blood sampling tube</td><td>Greiner Bio-One</td><td>VACUETTE 454217</td><td>Blood sampling &amp;amp; anticoagulating</td></tr><tr><td>Hematology analyzer</td><td>Siemens AG</td><td>ADVIA 2120i</td><td>Reference hematology analyzer</td></tr></tbody></table>

a rapid and chemical-free hemoglobin assay with photothermal angular light scattering

光熱角度光散乱（PT-AS）は、血液サンプルのヘモグロビン濃度（[Hbの]）を測定するための新規な光学的方法です。ヘモグロビン分子の固有光熱応答に基づいて、センサは高感度、[Hbの]の無農薬の測定を可能にします。 [ヘモグロビン] 0.35の範囲にわたって0.12グラム/ dlでの限界と検出能力 - 17.9グラム/ dlが以前に実証されています。この方法は、容易に、レーザポインタやウェブカメラなどの安価な消費者電子デバイスを使用して実施することができます。血液容器としてのマイクロキャピラリーチューブの使用はまた、ナノリットルスケールの血液量と低運転コストでヘモグロビン分析を可能にします。ここで、PT-AS光学装置と信号処理手順の詳細については、提示されています。実験プロトコルおよび貧血状態の血液サンプルについての代表的な結果（[ヘモグロビン] = 5.3、7.5、9.9グラム/デシリットル）も提供され、そして測定があちこちと比較しますミリアンペアの血液分析装置。実装と運用におけるそのシンプルさは、臨床検査室やリソースが制限された設定で、広い採用を有効にする必要があります。

ヘモグロビンアッセイは、PT-ASセンサを用いて行い、その測定値は、血液分析器からのものと比較しました。実験は2ヘルツの1.4 W / cm 2で、PTの変調周波数、及び5秒の測定時間のPTの励起光強度で行いました。 表1に実験条件をまとめたものです。プローブおよびPT励起光のビームサイズは、それぞれ、5.5および2mMでした。ウェブカメラは、30fpsのフ?...

Watch this Scientific Journal Video about A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering at JoVE.com

A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

光熱角度光散乱ラピッド・化学・フリーヘモグロビンアッセイ

Photo-thermal angular light scattering (PT-AS) is a novel optical method for measuring the hemoglobin concentration ([Hb]) of blood samples. On the basis ...

a-rapid-chemical-free-hemoglobin-assay-with-photothermal-angular

Research

JoVE Journal

Bioengineering

9.6K ビュー.  Yonsei University. A photo-thermal angular light scattering (PT-AS) sensor enables the rapid and chemical-free hemoglobin assay of nanoliter-scale blood samples. Here, details of the PT-AS setup and a measurement protocol for the hemoglobin concentration in blood are provided. Representative results for anemic blood samples are also presented.

ビデオ: A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering

Antigens Protected Functional Red Blood Cells By The Membrane Grafting Of Compact Hyperbranched Polyglycerols

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell anemia 1-3. Due to the presence of multitude of antigens on the RBC surface (~308 known antigens 4), patients in the chronic blood transfusion therapy develop alloantibodies due to the miss match of minor antigens on transfused RBCs 4, 5. Grafting of hydrophilic polymers such as polyethylene glycol (PEG) and hyperbranched polyglycerol (HPG) forms an exclusion layer on RBC membrane that prevents the interaction of antibodies with surface antigens without affecting the passage of small molecules such as oxygen ,glucose, and ions3. At present no method is available for the generation of universal red blood donor cells in part because of the daunting challenge presented by the presence of large number of antigens (protein and carbohydrate based) on the RBC surface and the development of such methods will significantly improve transfusion safety, and dramatically improve the availability and use of RBCs. In this report, the experiments that are used to develop antigen protected functional RBCs by the membrane grafting of HPG and their characterization are presented. HPGs are highly biocompatible compact polymers 6, 7, and are expected to be located within the cell glycocalyx that surrounds the lipid membrane 8, 9 and mask RBC surface antigens10, 11.

The cell membrane modification of red blood cells (RBCs) with hyperbranched polyglycerol (HPG) is presented. Modified RBCs were characterized by aqueous two phase partitioning, osmotic fragility and complement mediated lysis. The camouflage of surface proteins and antigens was evaluated using the flow cytometry and Micro Typing System (MTS) blood phenotyping cards.

抗原は、コンパクトな多分岐ポリグリセロールのグラフト膜によって機能赤血球を保護

Red blood cell (RBC) transfusion is vital for the treatment of a number of acute and chronic medical problems such as thalassemia major and sickle cell ...

免疫・感染学

A Rhodopsin Transport Assay by High-Content Imaging Analysis

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding disease that lacks effective treatment. We hypothesize that the cytotoxicity of the misfolded rhodopsin mutant can be alleviated by pharmacologically stabilizing the mutant rhodopsin protein. The P23H mutation, among the other Class II rhodopsin mutations, encodes a structurally unstable rhodopsin mutant protein that is accumulated in the endoplasmic reticulum (ER), whereas the wild type rhodopsin is transported to the plasma membrane in mammalian cells. We previously performed a luminescence-based high-throughput screen (HTS) and identified a group of pharmacological chaperones that rescued the transport of the P23H rhodopsin from ER to the plasma membrane. Here, using an immunostaining method followed by a high-content imaging analysis, we quantified the mutant rhodopsin protein amount in the whole cell and on the plasma membrane. This method is informative and effective to identify true hits from false positives following HTS. Additionally, the high-content image analysis enabled us to quantify multiple parameters from a single experiment to evaluate the pharmacological properties of each compound. Using this assay, we analyzed the effect of 11 different compounds towards six RP associated rhodopsin mutants, obtaining a 2-D pharmacological profile for a quantitative and qualitative understanding about the structural stability of these rhodopsin mutants and efficacy of different compounds towards these mutants.

Here, we described a high-content imaging method to quantify the transport of rhodopsin mutants associated with retinitis pigmentosa. A multiple-wavelength scoring analysis was used to quantify rhodopsin protein on the cell surface or in the whole cell.

高コンテンツ イメージング解析によるロドプシン輸送アッセイ

Rhodopsin misfolding mutations lead to rod photoreceptor death that is manifested as autosomal dominant retinitis pigmentosa (RP), a progressive blinding ...

化学

Rapid Determination of Antibody-Antigen Affinity by Mass Photometry

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this protocol, we describe the implementation of a new single-molecule technique, mass photometry (MP), for this purpose. MP is a label- and immobilization-free technique that detects and quantifies molecular masses and populations of antibodies and antigen-antibody complexes on a single-molecule level. MP analyzes the antigen-antibody sample within minutes, allowing for the precise determination of the binding affinity and simultaneously providing information on the stoichiometry and the oligomeric state of the proteins. This is a simple and straightforward technique that requires only picomole quantities of protein and no expensive consumables. The same procedure can be used to study protein-protein binding for proteins with a molecular mass larger than 50 kDa. For multivalent protein interactions, the affinities of multiple binding sites can be obtained in a single measurement. However, the single-molecule mode of measurement and the lack of labeling imposes some experimental limitations. This method gives the best results when applied to measurements of sub-micromolar interaction affinities, antigens with a molecular mass of 20 kDa or larger, and relatively pure protein samples. We also describe the procedure for performing the required fitting and calculation steps using basic data analysis software.

We describe a single-molecule approach to antigen-antibody affinity measurements using mass photometry (MP). The MP-based protocol is fast, accurate, uses a very small amount of material, and does not require protein modification.

質量光測定による抗体-抗原アフィニティーの迅速な測定

Measurements of the specificity and affinity of antigen-antibody interactions are critically important for medical and research applications. In this ...

生化学

Time-resolved F&#246;rster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to cytokines and growth factors. STAT proteins are activated by tyrosine phosphorylation mediated mainly by JAKs. The abnormal activation of STAT signaling pathways is implicated in many human diseases, especially cancer and immune-related conditions. Therefore, the ability to monitor STAT protein phosphorylation within the native cell signaling environment is important for both academic and drug discovery research. The traditional assay formats available to quantify phosphorylated STAT proteins include western blotting and the enzyme-linked immunosorbent assay (ELISA). These heterogeneous methods are labor-intensive, low-throughput, and often not reliable (specific) in the case of western blotting. Homogeneous (no-wash) methods are available but remain expensive. 
Here, detailed protocols are provided for the sensitive, robust, and cost-effective measurement in a 384-well format of endogenous levels of phosphorylated STAT1 (Y701), STAT3 (Y705), STAT4 (Y693), STAT5 (Y694/Y699), and STAT6 (Y641) in cell lysates from adherent or suspension cells using the novel THUNDER time-resolved F&#246;rster resonance energy transfer (TR-FRET) platform. The workflow for the cellular assay is simple, fast, and designed for high-throughput screening (HTS). The assay protocol is flexible, uses a low-volume sample (15 &#181;L), requires only one reagent addition step, and can be adapted to low-throughput and high-throughput applications. Each phospho-STAT sandwich immunoassay is validated under optimized conditions with known agonists and inhibitors and generates the expected pharmacology and Z'-factor values. As TR-FRET assays are ratiometric and require no washing steps, they provide much better reproducibility than traditional approaches. Together, this suite of assays provides new cost-effective tools for a more comprehensive analysis of specific phosphorylated STAT proteins following cell treatment and the screening and characterization of specific and selective modulators of the JAK/STAT signaling pathway.

Time-resolved F&#246;rster resonance energy transfer cell-based assay protocols are described for the simple, specific, sensitive, and robust quantification of endogenous phosphorylated signal transducer and activator of transcription (STAT) 1/3/4/5/6 proteins in cell lysates in a 384-well format.

ヒト細胞中の内因性リン酸化STATタンパク質の測定のための時間分解フェルスター共鳴エネルギー移動アッセイ

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to ...

Magnetic Levitation Coupled with Portable Imaging and Analysis for Disease Diagnostics

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for specialized equipment and trained personnel require that many diagnostic tests be performed at remote, centralized clinical laboratories. Magnetic levitation is a simple yet powerful technique and can be applied to levitate cells, which are suspended in a paramagnetic solution and placed in a magnetic field, at a position determined by equilibrium between a magnetic force and a buoyancy force. Here, we present a versatile platform technology designed for point-of-care diagnostics which uses magnetic levitation coupled to microscopic imaging and automated analysis to determine the density distribution of a patient's cells as a useful diagnostic indicator. We present two platforms operating on this principle: (i) a smartphone-compatible version of the technology, where the built-in smartphone camera is used to image cells in the magnetic field and a smartphone application processes the images and to measures the density distribution of the cells and (ii) a self-contained version where a camera board is used to capture images and an embedded processing unit with attached thin-film-transistor (TFT) screen measures and displays the results. Demonstrated applications include: (i) measuring the altered distribution of a cell population with a disease phenotype compared to a healthy phenotype, which is applied to sickle cell disease diagnosis, and (ii) separation of different cell types based on their characteristic densities, which is applied to separate white blood cells from red blood cells for white blood cell cytometry. These applications, as well as future extensions of the essential density-based measurements enabled by this portable, user-friendly platform technology, will significantly enhance disease diagnostic capabilities at the point of care.

We present a magnetic levitation technique coupled with automated imaging and analysis in both a smartphone-compatible device and a device with embedded imaging and processing. This is applied to measure the density distribution of cells with two demonstrated biomedical applications: sickle cell disease diagnosis and separating white and red blood cells.

病の診断のためのポータブルイメージングおよび分析と相まって磁気浮上

Currently, many clinical diagnostic procedures are complex, costly, inefficient, and inaccessible to a large population in the world. The requirements for ...

生物工学

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

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system. This system is constructed from a broadband white light source, a motorized filter wheel with a set of narrowband interference filters, a light guide, a collecting lens, a video zoom lens, and a monochromatic charged-coupled device (CCD) camera. An ellipsoidal cranial window is made in the skull bone of a rat under isoflurane anesthesia to capture in vivo multispectral diffuse reflectance images of the cortical surface. Regulation of the fraction of inspired oxygen using a gas mixture device enables the induction of different respiratory states such as normoxia, hyperoxia, and anoxia. A Monte Carlo simulation-based multiple regression analysis for the measured multispectral diffuse reflectance images at nine wavelengths (500, 520, 540, 560, 570, 580, 600, 730, and 760 nm) is then performed to visualize the two-dimensional maps of hemodynamics and the light scattering properties of the in vivo rat brain.

Simultaneous Evaluation of Cerebral Hemodynamics and Light Scattering Properties of the In Vivo Rat Brain Using Multispectral Diffuse Reflectance Imaging

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional multispectral diffuse reflectance imaging system.

脳血行動態との光散乱特性の同時評価<em&gt;インビボ</emマルチスペクトル拡散反射率イメージングの使用&gt;ラット脳

The simultaneous evaluation of cerebral hemodynamics and the light scattering properties of in vivo rat brain tissue is demonstrated using a conventional ...

神経科学

A Precision Medicine Tool for Measurement and Monitoring of Hemoglobin S in Sickle Cell Disease Patients Receiving Transfusion Therapy

Sickle cell disease (SCD) causes many severe health complications, including anemia, stroke, and acute chest syndrome. Red blood cell transfusion is the most commonly used therapy to treat or prevent these devastating complications. Other therapies include hydroxyurea therapy as well as bone marrow transplantation. Chronic intermittent transfusion is especially indicated to prevent recurrent strokes. However, transfusion therapy is associated with significant adverse effects (e.g., alloimmunization and iron overload). The point-of-care (POC) lateral flow immunoassay used here quantifies the %HbS in 15 min using a small patient blood sample. Utilizing this information, the healthcare provider is able to adjust blood transfusion volume for SCD patients to achieve the desired target HbS (most often &#60;30%), while reducing the risk of transfusion-related complications. When compared to laboratory hemoglobin electrophoresis data for 38 whole blood samples, the POC test performed with very high correlation and linear fit (slope = 0.9949, R2 = 0.9751). The strong agreement between the two hemoglobin S percentage (%HbS) quantifying methods shows that 89.5% of samples fall within &#177;5.2% HbS bias. The calibration for quantifying %HbS is built into the device to allow for an automated quantification of %HbS. This time- and cost-effective POC test thus allows the healthcare provider to make timely informed decisions when treating SCD patients, using accurate and updated data.

We have developed a point-of-care immunoassay to rapidly quantify hemoglobin S (HbS) levels during transfusion therapy for sickle cell disease (SCD) patients. By applying a small amount of treated blood to the device, the healthcare provider can determine the %HbS in a SCD patient to immediately inform clinical decisions.

Sickle cell disease (SCD) causes many severe health complications, including anemia, stroke, and acute chest syndrome. Red blood cell transfusion is the ...

医学

Homogeneous Time-resolved F&#246;rster Resonance Energy Transfer-based Assay for Detection of Insulin Secretion

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous insulin assays have existed for decades, the recent advent of homogeneous time-resolved F&#246;rster Resonance Energy Transfer (HTRF) technology has significantly simplified these measurements. This is a rapid, cost-effective, reproducible, and robust optical assay reliant upon antibodies conjugated to bright fluorophores with long lasting emission which facilitates time-resolved F&#246;rster Resonance Energy Transfer. Moreover, HTRF insulin detection is amenable for the development of high-throughput screening assays. Here we use HTRF to detect insulin secretion in INS-1E cells, a rat insulinoma-derived cell line. This allows us to estimate basal levels of insulin and their changes in response to glucose stimulation. In addition, we use this insulin detection system to confirm the role of dopamine as a negative regulator of glucose-stimulated insulin secretion (GSIS). In a similar manner, other dopamine D2-like receptor agonists, quinpirole, and bromocriptine, reduce GSIS in a concentration-dependent manner. Our results highlight the utility of the HTRF insulin assay format in determining the role of numerous drugs in GSIS and their pharmacological profiles.

Here, we present homogeneous time resolved FRET (HTRF) as an efficient method for rapid detection of insulin secreted from cells.

均質な時間分解蛍光共鳴エネルギー転送を用いた測定インスリン分泌の検出

The detection of insulin secretion is critical for elucidating mechanisms of regulated secretion as well as in studies of metabolism. Though numerous ...

<meta charset="utf8"></head><body>生体分子の VIS-SWIR 分光法の最適化

Characterization of Biological Absorption Spectra Spanning the Visible to the Short-Wave Infrared

For noninvasive light-based physiological monitoring, optimal wavelengths of individual tissue components can be identified using absorption spectroscopy. However, because of the lack of sensitivity of hardware at longer wavelengths, absorption spectroscopy has typically been applied for wavelengths in the visible (VIS) and near-infrared (NIR) range from 400 to 1,000 nm. Hardware advancements in the short-wave infrared (SWIR) range have enabled investigators to explore wavelengths in the ~1,000 nm to 3,000 nm range in which fall characteristic absorption peaks for lipid, protein, and water. These molecules are difficult to visualize in the VIS-NIR and can provide label-free sources of biological contrast. Furthermore, lower SWIR absorption has been observed for melanin, the primary chromophore responsible for skin pigmentation. In vivo&#160;optical devices like clinically standard pulse oximeters have been found to have reduced accuracy in people with darkly pigmented skin, possibly because of the stronger melanin absorption in the VIS range. Thus, error associated with skin pigmentation could be reduced by using devices operating in the SWIR. Optical instrument design is facilitated by the understanding of the absorption properties of core tissue components from the VIS to the SWIR range. This article describes protocols and instrumentation for obtaining VIS-SWIR absorption spectra of common tissue absorbers: oxygenated hemoglobin, deoxygenated hemoglobin, melanin, water, and lipid.

<meta charset="utf8"></head><body>可視から短波赤外までの生体吸収スペクトルの特性評価

Biological samples have distinct optical characteristics in the short-wave infrared (SWIR) compared to the visible (VIS) and near-infrared (NIR) wavelength ranges. However, recording pure SWIR absorption spectra is challenging and rarely conducted for biological molecules. This article presents a method to characterize the VIS-SWIR absorption spectra of biological absorbers.

可視から短波赤外までの生体吸収スペクトルの特性評価

For noninvasive light-based physiological monitoring, optimal wavelengths of individual tissue components can be identified using absorption spectroscopy. ...

光熱角度光散乱ラピッド・化学・フリーヘモグロビンアッセイ

要約

さらに動画を探す

この動画の章

抗原は、コンパクトな多分岐ポリグリセロールのグラフト膜によって機能赤血球を保護

高コンテンツイメージング解析によるロドプシン輸送アッセイ

質量光測定による抗体-抗原アフィニティーの迅速な測定

ヒト細胞中の内因性リン酸化STATタンパク質の測定のための時間分解フェルスター共鳴エネルギー移動アッセイ

病の診断のためのポータブルイメージングおよび分析と相まって磁気浮上

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

脳血行動態との光散乱特性の同時評価

A Precision Medicine Tool for Measurement and Monitoring of Hemoglobin S in Sickle Cell Disease Patients Receiving Transfusion Therapy

均質な時間分解蛍光共鳴エネルギー転送を用いた測定インスリン分泌の検出

可視から短波赤外までの生体吸収スペクトルの特性評価

光熱角度光散乱ラピッド・化学・フリーヘモグロビンアッセイ

要約

さらに動画を探す

この動画の章

抗原は、コンパクトな多分岐ポリグリセロールのグラフト膜によって機能赤血球を保護

高コンテンツ イメージング解析によるロドプシン輸送アッセイ

質量光測定による抗体-抗原アフィニティーの迅速な測定

ヒト細胞中の内因性リン酸化STATタンパク質の測定のための時間分解フェルスター共鳴エネルギー移動アッセイ

病の診断のためのポータブルイメージングおよび分析と相まって磁気浮上

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

脳血行動態との光散乱特性の同時評価

A Precision Medicine Tool for Measurement and Monitoring of Hemoglobin S in Sickle Cell Disease Patients Receiving Transfusion Therapy

均質な時間分解蛍光共鳴エネルギー転送を用いた測定インスリン分泌の検出

可視から短波赤外までの生体吸収スペクトルの特性評価

高コンテンツイメージング解析によるロドプシン輸送アッセイ