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Label-free biomarker detection from whole blood.

Stern E, Vacic A, Rajan NK, Criscione JM, Park J, Ilic BR, Mooney DJ, Reed MA, Fahmy TM - Nat Nanotechnol (2009)

Bottom Line: However, detecting these biomarkers in physiological fluid samples is difficult because of problems such as biofouling and non-specific binding, and the resulting need to use purified buffers greatly reduces the clinical relevance of these sensors.This two-stage approach isolates the detector from the complex environment of whole blood, and reduces its minimum required sensitivity by effectively pre-concentrating the biomarkers.This study marks the first use of label-free nanosensors with physiological solutions, positioning this technology for rapid translation to clinical settings.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, School of Engineering and Applied Science, Yale University, New Haven, Connecticut 06511, USA.

ABSTRACT
Label-free nanosensors can detect disease markers to provide point-of-care diagnosis that is low-cost, rapid, specific and sensitive. However, detecting these biomarkers in physiological fluid samples is difficult because of problems such as biofouling and non-specific binding, and the resulting need to use purified buffers greatly reduces the clinical relevance of these sensors. Here, we overcome this limitation by using distinct components within the sensor to perform purification and detection. A microfluidic purification chip simultaneously captures multiple biomarkers from blood samples and releases them, after washing, into purified buffer for sensing by a silicon nanoribbon detector. This two-stage approach isolates the detector from the complex environment of whole blood, and reduces its minimum required sensitivity by effectively pre-concentrating the biomarkers. We show specific and quantitative detection of two model cancer antigens from a 10 microl sample of whole blood in less than 20 min. This study marks the first use of label-free nanosensors with physiological solutions, positioning this technology for rapid translation to clinical settings.

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MPC operationa, Molecular structure of the photocleavable crosslinker. Primary antibody conjugation was performed with the amino group (right) and binding to chip-bound avidin occurred through the biotin group (left). b, Scanning electron micrograph of a representative w = 4 mm × l = 7 mm × h = 100 μm MPC capture-release chip. The inset is an optical image of MPC operation during washing. c, Schematic representation of PSA and CA15.3 detection using a modified ELISA technique. d, Fluorescence optical micrograph of an anti-OVA functionalized MPC following OVA-FITC-spiked whole blood flow and washing. The inset plots the pixel intensity (gray value, determined by ImageJ) versus position for the red cutline (green dataplot) and similar cutlines from images of post-UV irradiation and transfer (blue) and of an anti-PSA functionalized MPC following OVA-FITC-spiked blood flow and washing. The same exposure times were used for all images. e, Scatter plot showing the concentration of PSA and f, CA15.3 released from the MPC versus the concentration of PSA and CA15.3 introduced in whole blood, respectively. Each datapoint represents the average of three separate MPC runs and error bars represent one standard deviation.
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Figure 2: MPC operationa, Molecular structure of the photocleavable crosslinker. Primary antibody conjugation was performed with the amino group (right) and binding to chip-bound avidin occurred through the biotin group (left). b, Scanning electron micrograph of a representative w = 4 mm × l = 7 mm × h = 100 μm MPC capture-release chip. The inset is an optical image of MPC operation during washing. c, Schematic representation of PSA and CA15.3 detection using a modified ELISA technique. d, Fluorescence optical micrograph of an anti-OVA functionalized MPC following OVA-FITC-spiked whole blood flow and washing. The inset plots the pixel intensity (gray value, determined by ImageJ) versus position for the red cutline (green dataplot) and similar cutlines from images of post-UV irradiation and transfer (blue) and of an anti-PSA functionalized MPC following OVA-FITC-spiked blood flow and washing. The same exposure times were used for all images. e, Scatter plot showing the concentration of PSA and f, CA15.3 released from the MPC versus the concentration of PSA and CA15.3 introduced in whole blood, respectively. Each datapoint represents the average of three separate MPC runs and error bars represent one standard deviation.

Mentions: Figure 1 schematically illustrates the operation of the MPC chip. The avidin-functionalized chip19 (Fig. 1a) is treated with antibodies to any number of specific biomarkers conjugated to biotinylated, photocleavable crosslinkers containing a specific 19-mer DNA sequence (Fig. 2a)20. The MPC geometry was chosen to optimize biomarker binding (Supplementary Fig. S1)14 and chips were fabricated from 4-inch silicon wafers in a one-step photolithographic process (Supplementary Fig. S2). Completed chips (Fig. 2b) were loaded into a custom-machined flow chamber (inset, Fig. 2b and Supplementary Fig. S3), which enabled fluid handling and maintained a constant 5 μL volume in the system.


Label-free biomarker detection from whole blood.

Stern E, Vacic A, Rajan NK, Criscione JM, Park J, Ilic BR, Mooney DJ, Reed MA, Fahmy TM - Nat Nanotechnol (2009)

MPC operationa, Molecular structure of the photocleavable crosslinker. Primary antibody conjugation was performed with the amino group (right) and binding to chip-bound avidin occurred through the biotin group (left). b, Scanning electron micrograph of a representative w = 4 mm × l = 7 mm × h = 100 μm MPC capture-release chip. The inset is an optical image of MPC operation during washing. c, Schematic representation of PSA and CA15.3 detection using a modified ELISA technique. d, Fluorescence optical micrograph of an anti-OVA functionalized MPC following OVA-FITC-spiked whole blood flow and washing. The inset plots the pixel intensity (gray value, determined by ImageJ) versus position for the red cutline (green dataplot) and similar cutlines from images of post-UV irradiation and transfer (blue) and of an anti-PSA functionalized MPC following OVA-FITC-spiked blood flow and washing. The same exposure times were used for all images. e, Scatter plot showing the concentration of PSA and f, CA15.3 released from the MPC versus the concentration of PSA and CA15.3 introduced in whole blood, respectively. Each datapoint represents the average of three separate MPC runs and error bars represent one standard deviation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2818341&req=5

Figure 2: MPC operationa, Molecular structure of the photocleavable crosslinker. Primary antibody conjugation was performed with the amino group (right) and binding to chip-bound avidin occurred through the biotin group (left). b, Scanning electron micrograph of a representative w = 4 mm × l = 7 mm × h = 100 μm MPC capture-release chip. The inset is an optical image of MPC operation during washing. c, Schematic representation of PSA and CA15.3 detection using a modified ELISA technique. d, Fluorescence optical micrograph of an anti-OVA functionalized MPC following OVA-FITC-spiked whole blood flow and washing. The inset plots the pixel intensity (gray value, determined by ImageJ) versus position for the red cutline (green dataplot) and similar cutlines from images of post-UV irradiation and transfer (blue) and of an anti-PSA functionalized MPC following OVA-FITC-spiked blood flow and washing. The same exposure times were used for all images. e, Scatter plot showing the concentration of PSA and f, CA15.3 released from the MPC versus the concentration of PSA and CA15.3 introduced in whole blood, respectively. Each datapoint represents the average of three separate MPC runs and error bars represent one standard deviation.
Mentions: Figure 1 schematically illustrates the operation of the MPC chip. The avidin-functionalized chip19 (Fig. 1a) is treated with antibodies to any number of specific biomarkers conjugated to biotinylated, photocleavable crosslinkers containing a specific 19-mer DNA sequence (Fig. 2a)20. The MPC geometry was chosen to optimize biomarker binding (Supplementary Fig. S1)14 and chips were fabricated from 4-inch silicon wafers in a one-step photolithographic process (Supplementary Fig. S2). Completed chips (Fig. 2b) were loaded into a custom-machined flow chamber (inset, Fig. 2b and Supplementary Fig. S3), which enabled fluid handling and maintained a constant 5 μL volume in the system.

Bottom Line: However, detecting these biomarkers in physiological fluid samples is difficult because of problems such as biofouling and non-specific binding, and the resulting need to use purified buffers greatly reduces the clinical relevance of these sensors.This two-stage approach isolates the detector from the complex environment of whole blood, and reduces its minimum required sensitivity by effectively pre-concentrating the biomarkers.This study marks the first use of label-free nanosensors with physiological solutions, positioning this technology for rapid translation to clinical settings.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, School of Engineering and Applied Science, Yale University, New Haven, Connecticut 06511, USA.

ABSTRACT
Label-free nanosensors can detect disease markers to provide point-of-care diagnosis that is low-cost, rapid, specific and sensitive. However, detecting these biomarkers in physiological fluid samples is difficult because of problems such as biofouling and non-specific binding, and the resulting need to use purified buffers greatly reduces the clinical relevance of these sensors. Here, we overcome this limitation by using distinct components within the sensor to perform purification and detection. A microfluidic purification chip simultaneously captures multiple biomarkers from blood samples and releases them, after washing, into purified buffer for sensing by a silicon nanoribbon detector. This two-stage approach isolates the detector from the complex environment of whole blood, and reduces its minimum required sensitivity by effectively pre-concentrating the biomarkers. We show specific and quantitative detection of two model cancer antigens from a 10 microl sample of whole blood in less than 20 min. This study marks the first use of label-free nanosensors with physiological solutions, positioning this technology for rapid translation to clinical settings.

Show MeSH
Related in: MedlinePlus