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

혈액 샘플 실험은 관련 법률 및 제도적 지침에 따라 수행 하였다. 샘플은 인수 기관에서 임상 시험에서 처리했다 잔류 혈액 샘플을했다. 1. PT-AS 광 설치 참고 : 하나는 초기 PT-AS 설정에 빈 마이크로 모세관 튜브를 사용할 수 있습니다. <ol><li> 각각 200 및 330 μm의의 내부 및 외부 직경을 갖는 빈 마이크로 모세관 튜브 및 모세관 고정구에 큰 ~ 5 cm 이상의 길이를 탑재. 시판 섬유 일정 튜브 치구로서 사용할 수있다. </li><li> 안전하게 모세관을 조명하는 광원을 조사, 즉, 650 nm의 레이저 포인터를 고정. 프로브 빔이 모세관보다 커야한다. 각도주기적인 패턴을 관찰하는 모세관 뒤에 화면 (예 : 흰색 종이)를 놓습니다. </li><li> 검출부를 들어, 직접 scatt 캡처 웹캠 어떠한 렌즈를 제거패턴를 끄기. 프로브 빔 방향에 대해 25 ~ 35 °의 각도에서 모세관 뒤에 웹캠 배치. 모세관에 의해 생성 된 각주기 패턴 검출기 (도 1)에 의해 측정 될 수 있음을 보장한다. 이미지 센서가 위치 될 때, 이미지 센서의 중간 각도주기 패턴을 관찰한다. </li><li> 모세관을 조명 할 수있는 532 나노 PT의 여기 광원을 배치합니다. 만큼 PT 여기 광이 모세관 튜브 프로브 빔과 중첩 직접 검출기에 도달하지 않는 한, 임의의 각도로 PT 광원을 배치. 는 RI에 큰 변화를 초래으로 일반적으로 높은 광 출력을 이용하여 혈액 샘플의 PT 여기는 PT-AS 감도를 향상시킨다. <ol><li> 채용 된 PT 여기 광원에 가장 높은 광 출력을 사용한다. 또한, PT 여기 광이 모세관에 탐침 광을 중첩되도록. 태평양 표준시 여기 광의 빔의 크기를 사용하여배 이상의 광 프로브의 전체 프로브 체적을 가열. </li></ol></li><li> 532 nm의 광을 차단하고 단지 650 nm의 프로브 광을 측정하는 검출기 앞에 긴 패스 필터를 배치했다. </li><li> 모세관을 조사하기 전에 PT 여기 광의 경로에 광 초퍼를 설치한다. 광 초퍼는 PT의 여기 광 강도를 변조하는데 사용된다. </li></ol> 2. 혈액 샘플 준비 <ol><li> 에틸렌 디아민 테트라 아세트산 혈액 샘플링 튜브에 빈혈 상태에 신선한 전혈 6 ml의 그리기, 잘 샘플을 섞는다. 다른 처리가 필요하지 않습니다. </li><li> 혈액 응고를 방지하는 추출 24 시간 안에 PT-AS 센서를 이용하여 혈액 시료를 측정한다. </li></ol> 3. PT-AS 측정 프로토콜 <ol><li> 측정하기위한 혈액 샘플을 마이크로 모세관을로드. 혈액의 튜브에 배치하여 모세관 작용을 통해 혈액 모세관 채우기충분한. 측정에 필요한 최소 샘플 량은 모세관의 내경 및 프로브 빔의 크기에 의해 결정된다. <ol><li> 200 μm의 내경을 가진 튜브를 이용한다. 프로브 빔 사이즈 측정이&gt; 63 NL 샘플 부피로 수행 될 수 있음을 시사 대표적인 결과 2 mm였다. </li></ol></li><li> 고정구에있는 지정된 위치에있는 모세관 튜브를 탑재합니다. </li><li> 혈액이 장착 된 마이크로 모세관 튜브를 조명하기 위해 650 나노 프로브 레이저를 켭니다. 각도주기적인 패턴은 웹캠으로 관찰해야한다. </li><li> 튜브를 조명하기 위해 532 나노 PT 여기 레이저를 켭니다. </li><li> 2 Hz에서의 PT 여기 광의 강도를 변조하는 광 초퍼를 실행. 주 :이 운전 조건의 선택을위한 이론적 근거는 토론 및 Kim 등으로 설명된다. 12. <ol><li> 광 초퍼 모터 헤드 어셈블리 초퍼 휠 장착체계. </li><li> 헬기 컨트롤 박스의 전원을 켜고 변조 주파수를 설정 콘솔의 컨트롤 노브를 사용합니다. </li><li> 컨트롤 노브를 사용하여 헬기를 실행합니다. </li></ol></li><li> MPEG-4 (MP4) 형식으로 5 초 동안 웹캠을 통해 변동 산란 패턴을 기록합니다. </li></ol> 4. 신호 처리 주 : PT-AS 신호 처리 실험실 개발 MATLAB 코드를 사용하여 수행 하였다. <ol><li> 이미지를 추출 할 동영상 파일을로드합니다. 각 화상의 세로 방향의 화소 값의 평균을 계산하여 평균 산란 패턴을 얻었다 [대표 이미지도 2 (a)를 참조]도 2 (b, c)]. </li><li> 푸리에 평균 산란 패턴의 변형 평가하고 최고 공간 주파수에서의 위상을 계산한다. 모든 녹화 된 영상의 모든 프레임에 대해 이러한 작업을 수행합니다. </li><li> 모든 이미지로부터 획득 된 위상 값을 이용하여, 시간 상 플롯변동 [그림 2의 (d)]. 위상이 PT 변조 주파수 변동합니다. 푸리에 변조 주파수의 크기를 시간 영역에서의 위상 변동 변환 및 수득보십시오. 이 신호는 PT-AS 신호라고한다 [도 2의 (e)]. </li><li> 내로의 PT-AS 신호를 전환하여 혈액 샘플의 [헤모글로빈]을 측정 프로토콜 (5)에서 얻어진 검량선을 이용하여 헤모글로빈] 대응. </li></ol> 5. PT-AS 교정 <ol><li> (- 18g / DL 예컨대, 0)의 Hb] 균일 PT-AS 센서의 검출 범위에 분포 값을 갖는, 혈액 샘플을 준비한다. </li><li> 보정 전, 기준 혈액 분석기를 사용하여 샘플의 [헤모글로빈] 값을 정량화. 샘플의 PT-AS 신호를 측정한다. </li><li> experimenta의 교정 최소 제곱 적합 선형를 수행하여 PT-AS 신호 [헤모글로빈] 관련 곡선 [헤모글로빈] = A [PT-AS 신호] + B를 도출리터 결과. 표 1에 규정 된 작동 조건 [헤모글로빈] 및 PT-AS 신호 사이의 관계는 [헤모글로빈]였다 = 5.13 [PT-AS 신호] - 0.09. 선형 피팅을 수행하기 위해 MATLAB 코드를 사용합니다. </li></ol>

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No conflict of interest is declared.

PT-AS 센서는 처리되지 않은 혈액 샘플을 직접 [헤모글로빈] 측정 할 수있는 모든 광학 방법을 나타낸다. 상기 방법은 적혈구의 헤모글로빈 분자의 고유 PT 응답하여 혈중 [헤모글로빈] 정량화한다. 532 nm의 빛에 의해 조명 아래에서, 헤모글로빈 분자는 빛 에너지를 흡수하고 열을 생산하고 있습니다. 얻어진 온도 상승은 혈액 샘플의 RI를 변화시킨다. BSI의 높은 RI 감도는 혈액에서이 RI의 변화를 측정...

혈액 검사는 일반적으로 전체 인간의 건강을 평가하는 특정 질환에 관련 바이오 마커를 검출하기 위해 수행된다. 예를 들어, 혈중 콜레스테롤 농도는 밀접한 심혈관 질환 및 췌장염 관련되어 고지혈증의 기준으로서 역할을한다. 혈당 수준이 당뇨병 성 케톤 산증과 고혈당 삼투압 증후군과 관련된 합병증으로 혈당 내용은 자주 측정해야한다. 말라리아, 인간 면역 결핍 바이러스와 후천성 면역 결핍 증후군 같은 심각한 질병을 혈액 검사에 의해 진단하고, 적혈구, 혈소판을 포함한 혈액 성분의 정량 및 백혈구 췌장 및 신장 질환의 스크리닝을 가능하게한다. 헤모글로빈 (혈색소), 혈액의 중요한 구성 요소는 적혈구의 약 96 %를 구성하고, 사람의 장기에 산소를 수송한다. 질량 농도의 중요한 변경 ([혈색소])는 저를 나타낼 수있다tabolic 변경, 간담 질환,과, 신경 심혈관 및 내분비 장애 1. [혈색소 따라서 정기적으로 혈액 검사로 측정됩니다. 특히, 빈혈 환자, 투석 환자, 임산부에 강하게 중요한 작업이 같이 헤모글로빈]을 모니터링하는 것이 좋습니다. 다양한 [헤모글로빈] 검출 방법에 따라서 개발되었다. 헤모글로빈 시안화 방법 [헤모글로빈] 정량 가장 흔한 방법 중 하나는, 시안화 칼륨 (KCN)은 적혈구 (3)의 지질 이중층을 파괴하기 위해 사용한다. 540 nm의 주위 화학 나타낸다 높은 흡수에 의해 생성 된 시안 헤모글로빈; 따라서, [혈색소] 측정은 비색 분석을 통해 할 수있다. 이 방법은 널리 단순성 때문에 사용되지만 사용되는 화학 물질 (예, KCN 및 dimethyllaurylamine 산화물)은 인간과 환경에 독성이있다. 헤마토크릿 방식은 전체 혈액의 부피에 비하여 적혈구의 체적비를 측정원심 분리를 통해 매화; 그러나 비교적 큰 혈액량 (50-100 μL) (4)를 필요로한다. 측정법 방법의 측정은 [헤모글로빈] 정확히 어떤 화학 물질이없는,하지만 여러 파장과 큰 혈액량의 측정은 5,6 필요합니다. 유사하게, [헤모글로빈] 광학 측정을위한 여러 방법이 광 산란에 기초하여 검출 방법을 포함하는 제안되어 있지만, 그 측정 정밀도는 혈액 이론적 모델의 정확도에 강하게 의존한다. 이러한 한계를 극복하기 위해, 헤모글로빈의 광열 (PT) 효과에 기초하여 헤모글로빈] 검출 방법은 최근 7이 제안되어있다. 주로 산화철로 구성되어 혈색소는 532 nm에서 빛을 흡수하고 열 8-10로 빛 에너지를 변환합니다. 이 PT의 온도 상승은 혈액 시료의 굴절률 (RI)의 변화를 측정함으로써 광학적으로 검출 할 수있다. 임 등. 고용 스펙트럼 영역 빛 간섭 reflectometrY는 피 수용 실 (11) 내의 PT 광로 길이의 변화를 측정한다. 상기 방법은 무공해 직접 [헤모글로빈] 측정 가능하지만, 분광기의 사용과 간섭계 장치는 소형화를 저해 할 수있다. 최근 대안 [헤모글로빈] 검출 방법을 제시 장치 (12)의 소형화에 더 적합 광열 각도 광산란 (PT-AS) 센서를 불린다. PT-AS 센서 모세관 안에 혈액 샘플의 RI PT의 변화를 측정하는 백 산란 간섭계 (BSI)의 RI 높은 감도를 이용한다. BSI 다양한 솔루션 13-15의 RI를 측정하는 자유 용액 16 생화학 적 상호 작용을 모니터링하는 데 이용되어왔다. 태평양 표준시-AS 센서는 BSI와 같이 유사한 광학 장치를 사용하지만, 혈액 샘플에서 RI의 PT 증가를 측정하는 광열 여기 설정을 결합한 제품입니다. BSI에 상기 PT-AS 센서의 작동 원리는 다른 곳에서 상세하게 설명된다 <sup&gt; 12,15. PT-AS 센서는 넓은 감지 범위 (0.35-17.9 g / DL)을 통해 고감도 [혈색소] 측정을 입증하고 &lt;100 NL의 샘플 볼륨으로 운영 할 수있다. 혈액 샘플의 어떤 전처리가 불필요하며, 측정 시간은 5 초 ~하다. 여기서, 실험 장치 상세한 측정 프로토콜이 설명된다. 주제 PT-AS 결과 빈혈 환자의 혈액 샘플을 이용하여 제공되고, 그 결과는 PT-AS 센서의 정확도를 평가하기 위하여 혈액 분석 장치에서 그와 비교된다.

<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

광열 각도 광산란 (PT-AS)은 혈액의 헤모글로빈 농도 ([헤모글로빈])를 측정하는 신규 한 광학 방법이다. 헤모글로빈 분자의 고유 광열 응답에 기초하여, 센서는 고감도 [헤모글로빈]의 무농약 측정을 가능하게한다. [혈색소] 0.35의 범위에서 0.12 g / DL의 한계와 검색 기능 - 17.9 g / DL는 이전에 증명되었다. 이 방법은 용이하게 이러한 레이저 포인터 웹캠으로 저렴 가전 기기를 이용하여 구현 될 수있다. 혈액 용기와 같은 마이크로 모세관 튜브의 사용은 또한 나노 리터 규모 혈액량과 낮은 운전 비용으로 헤모글로빈 분석을 가능하게한다. 여기서, PT-AS 광 설치 및 신호 처리 절차에 대한 자세한 내용은되게됩니다. 실험 프로토콜 및 빈혈 상태에서 혈액 샘플 대표적인 결과 ([헤모글로빈 = 5.3, 7.5 및 9.9 g / DL)도 제공되고, 측정은 그 이리저리 비교엄마의 혈액 분석기. 구현 및 운영에 단순 임상 실험실 및 자원 제한 설정에서 넓은 채택을 사용하도록 설정해야합니다.

헤모글로빈 분석은 PT-AS 센서를 이용하여 수행하였고, 그 측정은 혈액 분석 장치에서 비교 하였다. 실험은 1.4 W / cm 2 초, 5, PT 변조 2 Hz의 주파수 및 측정 시간 PT 여기 광 강도로 하였다. 표 1은 실험 조건을 요약한다. 프로브 및 PT 여기 광의 빔의 크기는 각각 5.5 및 2mm이었다. 웹캠 30 fps의 프레임 율로 이미지를 기록했다. 측정을 위해, 세 가지의 Hb 농도 ?...

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.

광열 각도 광산란와 신속하고 화학 무료 헤모글로빈 분석

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

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Research

JoVE Journal

Bioengineering

9.6K 조회수.  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.

비디오: 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.

항원은 컴팩트 Hyperbranched Polyglycerols 중 이식 막으로 기능 적혈구를 보호

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 ...

연구

면역학 및 감염병학

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.

이 콘텐츠 이미징 분석 Rhodopsin 전송 분석 결과

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

화학

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.

질량 광합성에 의한 항체 항원 선호도의 신속한 결정

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

생화학

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.

인간 세포에서 내인성 인산화 STAT 단백질의 측정을 위한 시간 분해된 Förster 공명 에너지 전달 분석

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.

질병 진단을위한 휴대용 이미징 및 분석과 결합 자기 부상

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

생체공학

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.

자기 변조 Biosensing 시스템을 사용하여 생물 학적 Assays의 급속한 동차 검색

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

생물학

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.

대뇌 혈류와의 광 산란 특성을 동시에 평가<em&gt; 생체</em&gt; 다중 스펙트럼 확산 반사 이미징을 이용한 쥐 뇌

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

신경과학

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 ...

의학

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.

균질 시간 해결 포스터 공명 에너지 전송 기반 분석 결과 인슐린 분 비의 검출에 대 한

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>생물학적 분자를 위한 VIS-SWIR 분광법 최적화

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>Realistic Absorption Spectra Spantra Spanling Spaning the Visible to the Short-Wave Infrared(생물학적 흡수 스펙트럼의 특성화: 가시광선에서 단파 적외선까지)

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.

Realistic Absorption Spectra Spantra Spanling Spaning the Visible to the Short-Wave Infrared(생물학적 흡수 스펙트럼의 특성화: 가시광선에서 단파 적외선까지)

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